Abstract: The polar ocean presently surrounding Antarctica is the coldest, most thermally stable marine environment on earth. Because oxygen solubility in seawater is inversely proportional to temperature, the cold Antarctic seas are an exceptionally oxygen-rich aquatic habitat. Eight families of a single perciform suborder, the Notothenioidei, dominate the present fish fauna surrounding Antarctica. Notothenioids account for approximately 35% of fish species and 90% of fish biomass south of the Antarctic Polar Front. Radiation of closely related notothenioid species thus has occurred rapidly and under a very unusual set of conditions: relative oceanographic isolation from other faunas due to circumpolar currents and deep ocean trenches surrounding the continent, chronically, severely cold water temperatures, very high oxygen availability, very low levels of niche competition in a Southern Ocean depauperate of species subsequent to a dramatic crash in species diversity of fishes that occurred sometime between the mid-Tertiary and present. These features make Antarctic notothenioid fishes an uniquely attractive group for the study of physiological and biochemical adaptations to cold body temperature. Few distinctive features of Antarctic fishes are as unique as the pattern of expression of oxygen-binding proteins in one notothenioid family, the Channichthyidae (Antarctic icefishes). All channichthyid icefishes lack the circulating oxygen-binding protein, hemoglobin (Hb); the intracellular oxygen-binding protein, myoglobin (Mb) is not uniformly expressed in species of this family. Both proteins are normally considered essential for adequate delivery of oxygen to aerobically poised tissues of animals. To compensate for the absence of Hb, icefishes have developed large hearts, rapidly circulate a large blood volume and possess elaborate vasculature of larger lumenal diameter than is seen in red-blooded fishes. Loss of Mb expression in oxidative muscles correlates with dramatic elevation in density of mitochondria within the cell, although each individual organelle is less densely packed with respiratory proteins. Within the framework of oxygen movement, the adaptive significance of greater vascular density and mitochondrial populations is understandable but mechanisms underlying development of these characteristics remain unknown. The answer may lie in another major function of both Hb and Mb, degradation of the ubiquitous bioactive compound, nitric oxide (NO). The research will test the hypothesis that loss of hemoprotein expression in icefishes has resulted in an increase in levels of NO that mediate modification of vascular systems and expansion of mitochondrial populations in oxidative tissues. The objectives of the proposal are to quantify the vascular density of retinas in +Hb and -Hb notothenioid species, to characterize NOS isoforms and catalytic activity in retina and cardiac muscle of Antarctic notothenioid fishes, to evaluate level of expression of downstream factors implicat ed in angiogenesis (in retinal tissue) and mitochondrial biogenesis (in cardiac muscle), and to determine whether inhibition of NOS in vivo results in regression of angiogenic and mitochondrial biogenic responses in icefishes. Broader impacts range from basic biology, through training of young scientists, to enhanced understanding of clinically relevant biomedical processes.