The blood of Antarctic icefishes (family Channichthyidae, suborder Notothenioidei) is completely devoid of hemoglobin. Icefishes have developed compensatory adaptations that reduce oxygen demand and enhance oxygen transport. Oxygen delivery to tissues occurs by carrying the gas physically dissolved in the plasma. To evaluate the evolutionary pathway leading to the icefish hemoglobinless phenotype, the adult and embryonic/juvenile gene complexes from a closely related, red-blooded notothenioid species were isolated and characterized. The hybridization pattern of notothenioid adult globin cDNAs showed that the genomes of three icefish species retain transcriptionally inactive alpha1-globin-related DNA sequences, which are identical truncated variants of the alpha1-globin gene of the red-blooded fish, containing part of intron 2, all of exon 3, and the 3'-untranslated region. The icefish genomes have no beta-globin genes. Furthermore, Southern blots of genomic DNA from red- and white-blooded (two species) notothenioids, probed with fragments of the genes flanking the ends of the embryonic/juvenile complex, indicated that icefishes have also lost embryonic/juvenile globin genes. It is proposed that inability to express hemoglobin arose from a single, large-scale deletional event, which removed all icefish globin genes with the exception of the 3' end of alpha1.