Abstract The rates and patterns of molecular evolution in many eukaryotic organisms have been shown to be influenced by the compartmentalization of their genomes into fractions of distinct base composition and mutational properties. We have examined the Drosophila genome to explore relationships between the nucleotide content of large chromosomal segments and the base composition and rate of evolution of genes within those segments. Direct determination of the G + C contents of yeast artificial chromosome clones containing inserts of Drosophila melanogaster DNA ranging from 140-340 kb revealed significant heterogeneity in base composition. The G + C content of the large segments studied ranged from 36.9% G + C for a clone containing the hunchback locus in polytene region 85, to 50.9% G + C for a clone that includes the rosy region in polytene region 87. Unlike other organisms, however, there was no significant correlation between the base composition of large chromosomal regions and the base composition at fourfold degenerate nucleotide sites of genes encompassed within those regions. Despite the situation seen in mammals, there was also no significant association between base composition and rate of nucleotide substitution. These results suggest that nucleotide sequence evolution in Drosophila differs from that of many vertebrates and does not reflect distinct mutational biases, as a function of base composition, in different genomic regions. Significant negative correlations between codon-usage bias and rates of synonymous site divergence, however, provide strong support for an argument that selection among alternative codons may be a major contributor to variability in evolutionary rates within Drosophila genomes.