Hypoxia-inducible factor (HIF) is a crucial regulator of cellular and systemic responses to low oxygen levels. Here we firstly cloned three HIF-alpha isoforms from the basal branches of Osteichthyes and used computational tools to characterise the molecular change underlying the functional divergence of HIF-alpha isoforms in different lineages. Only the HIF-1 alpha and HIF-2 alpha in African lungfish and amphibians were found under positive selection. HIF-1 alpha and -2 alpha were less functionally divergent in basal ray-finned fish than in teleosts, and showed conserved but different transcriptional activity towards specific target genes. (C) 2013 The Authors, Published by Elsevier B.V. on behalf of Federation of European Biochemical Societies. All rights reserved.