A method for estimating the evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences is presented. This method is applied to genes of phi X174 and G4 genomes, histone genes and beta-globin genes, for which homologous nucleotide sequences are available for comparison to be made. It is shown that the rates of synonymous substitutions are quite uniform among the non-overlapping genes of phi X174 and G4 and among histone genes H4, H2B, H3 and H2A. A comparison between phi X174 and G4 reveals that, in the overlapping segments of the A-gene, the rate of synonymous substitution is reduced more significantly than the rate of amino acid substitution relative to the corresponding rate in the non-overlapping segment. It is also suggested that, in the coding region surrounding the splicing points of intervening sequences of beta-globin genes, there exist rigid secondary structures. It is in only these regions that the beta-globin genes show the slowing down of evolutionary rates of both synonymous and amino acid substitutions in the primate line.