Previous comparison of a relatively small set of homologous genes from Escherichia coli and Salmonella typhimurium revealed that genes nearer to the origin of replication had substitution rates lower than genes closer to the replication terminus. The recently completed sequences of numerous bacterial genomes have allowed us to test whether this effect of distance from the replication origin on substitution rates, as observed for the E. coli-S. typhimurium comparison, is a general feature of bacterial genomes. Extending the analysis to all 3,000 E. coli-S. typhimurium homologs confirmed the significant association between chromosomal position and synonymous site divergence. However, the effect, though still significant, is not as dramatic as originally thought. A similar association between relative chromosomal location and synonymous substitution rate was detected in the majority of other bacterial species comparisons within alpha- and gamma- Proteobacteria, and Firmicutes but was absent in Chlamydiales. The opposite trend, i.e., a decrease in synonymous divergence with distance from the replication origin, was detected in Mycobacteria. Analysis of the patterns of nucleotide substitutions revealed that the distance effect is not affected by gene orientation and is mainly caused by an increase in rates of transversions, suggesting that this effect may not be caused by recombinational repair or biased gene conversion, as originally suggested.