Buchnera, the bacterial endosymbionts of aphids, undergo severe population bottlenecks during maternal transmission through their hosts. Previous studies suggest an increased effect of drift within these strictly asexual, small populations, resulting in an increased fixation of slightly deleterious mutations. This study further explores sequence evolution in Buchnera using three approaches. First, patterns of codon usage were compared across several homologous Escherichia coli and Buchnera loci, in order to test the prediction that selection for the use of optimal codons is less effective in small populations. A chi 2-based measure of codon bias was developed to adjust for the overall A + T richness of silent positions in the endosymbionts. In contrast to E. coli homologues, adaptive codon bias across Buchnera loci is markedly low, and patterns of codon usage lack a strong relationship with gene expression level. These data suggest that codon usage in Buchnera has been shaped largely by mutational pressure and drift rather than by selection for translational efficiency. One exception to the overall lack of bias is groEL, which is known to be constitutively overexpressed in Buchnera and other endosymbionts. Second, relative-rate tests show elevated rates of sequence evolution of numerous protein-coding loci across Buchnera, compared to E. coli. Finally, consistently higher ratios of nonsynonymous to synonymous substitutions in Buchnera loci relative to the enteric bacteria strongly suggest the accumulation of nonsynonymous substitutions in endosymbiont lineages. Combined, these results suggest a decreased effectiveness of purifying selection in purging endosymbiont populations of slightly deleterious mutations, particularly those affecting codon usage and amino acid identity.