Synonymous codons are not used at random, significantly influencing the base composition of the genome. The selection-mutation-drift model proposes that this bias reflects natural selection in favor of a subset of preferred codons. Previous estimates in Drosophila of the intensity of selective forces involved seem too large to be reconciled with theoretical predictions of the level of codon bias. This probably results from confounding effects of the demographic histories of the species concerned. We have studied three species of the virilis group of Drosophila, which are more likely to satisfy the assumptions of the evolutionary models. We analyzed the patterns of polymorphism and divergence in a sample of 18 genes and applied a new method for estimating the intensity of selection on synonymous mutations based on the frequencies of unpreferred mutations among polymorphic sites. This yielded estimates of selection intensities (N(e)s) of the order of 0.65, which is more compatible with the observed levels of codon bias. Our results support the action of both selection and mutational bias on codon usage bias and suggest that codon usage and genome base composition in the D. americana lineage are in approximate equilibrium. Biased gene conversion may also contribute to the observed patterns.