The icefishes of the Southern Ocean (family Channichthyidae, suborder Notothenioidei) are unique among vertebrates in their inability to synthesize hemoglobin. We have shown previously (Cocca, E., Ratnayake-Lecamwasam, M., Parker, S. K., Camardella, L., Ciaramella, M., di Prisco, G., and Detrich, H. W., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1817-1821) that icefishes retain inactive genomic remnants of adult notothenioid ␣-globin genes but have lost the gene that encodes adult ␤-globin. Here we demonstrate that loss of expression of the major adult ␣-globin, ␣1, in two species of icefish (Chaenocephalus aceratus and Chionodraco rastrospinosus) results from truncation of the 5 end of the notothenioid ␣1-globin gene. The wild-type, functional ␣1-globin gene of the Antarctic yellowbelly rockcod, Notothenia coriiceps, contains three exons and two A ؉ T-rich introns, and its expression may be controlled by two or three distinct promoters. Retained in both icefish genomes are a portion of intron 2, exon 3, and the 3-untranslated region of the notothenioid ␣1-globin gene. The residual, nonfunctional ␣-globin gene, no longer under positive selection pressure for expression, has apparently undergone random mutational drift at an estimated rate of 0.12-0.33%/million years. We propose that abrogation of hemoglobin synthesis in icefishes most likely resulted from a single mutational event in the ancestral channichthyid that deleted the entire ␤-globin gene and the 5 end of the linked ␣1-globin gene.