Unlike temperate fishes, Antarctic fishes of the notothenioid suborder, whose body temperatures (−2 to 1°C) conform to the Southern Ocean, must express their genomes in an extremely cold thermal regime. To determine whether these fishes have evolved compensatory adjustments that maintain efficient gene transcription at low temperatures, we have initiated studies of the cis-acting regulatory elements that control globin gene expression in the Antarctic rockcod Notothenia coriiceps and in its close relative, the temperate New Zealand black cod N. angustata (habitat temperature = 6 to 15°C). The genes encoding the major α1 and β globins of these fishes are tightly linked in head-to-head (5′ to 5′) orientation. The intergenic regions separating the globin genes in the two fishes, ∼4.3 kb in N. coriiceps and ∼3.2 kb in N. angustata, are highly similar in sequence, the major difference being the absence of a 1.1-kb, repeat-containing segment in the latter. To assess the promoter and enhancer activities of the intergenic regions, each was cloned into the luciferase-reporter vector pGL3-Basic, and the constructs were transfected into MEL cells. Upon DMSO induction of MEL cell differentiation, each of the α/β-intergenic regions functioned in both orientations as erythroid-responsive transcriptional regulators. However, expression of luciferase mediated by the N. coriiceps intergene was 6-fold greater in the α orientation than that for the N. angustata intergene and 2-fold greater for the β. The greater transcription-stimulating activity of the N. coriiceps intergene can be attributed to two enhancers composed of combinations of CAC/Sp1 and GATA motifs and located in direct repeat elements. N. angustata, which lacked repetitive structure in its intergene, contained a single copy of the enhancer. We propose that cold adaptation of globin gene expression in N. coriiceps evolved in part through duplication and refinement of critical cis-acting regulatory elements as the Southern Ocean cooled during the past 25 million years.