Molecular evolution of the histone multigene family was studied by cloning and sequencing regions of the histone 3 gene in the Drosophila melanogaster species subgroup. Analysis of the nucleotide substitution pattern showed that in the coding region synonymous changes occurred more frequently to A or T in contrast to the CC-rich base composition, while in the 3' region the nucleotide substitutions were most likely in equilibrium. These results suggested that the base composition at the third codon position of the H3 gene, i,e,, codon usage, has been changing to A or T in the Drosophila melanogaster species subgroup, (C) 2000 Academic Press.