A phylogenetic analysis of mammalian type I interferon (IFN) genes showed: (1) that the three main subfamilies of these genes in mammals (IFN-beta, IFN-alpha, and IFN-omega) diverged after the divergence of birds and mammals but before radiation of the eutherian orders and (2) that IFN-beta diverged first. Although apparent cases of interlocus recombination among mouse IFN-alpha genes were identified, the hypothesis that coding regions of IFN-alpha genes have been homogenized within species by interlocus recombination was not supported. Flanking regions as well as coding regions of IFN-alpha were more similar within human and mouse than between these species; and reconstruction of the pattern of nucleotide substitution in IFN-alpha coding regions of four mammalian species by the maximum parsimony method suggested that parallel substitutions have occurred far more frequently between species than within species. Therefore, it seems likely that IFN-alpha genes have duplicated independently within different eutherian orders. In general, type I IFN genes are subject to purifying selection, which in the case of IFN-alpha and IFN-beta is strongest in the putative receptor-binding domains. However, analysis of the pattern of nucleotide substitution among IFN-omega genes suggested that positive Darwinian selection may have acted in some cases to diversify members of this subfamily at the amino acid level.