The primary endosymbionts of aphids are maternally inherited bacteria that live only within specialized host cells. Phylogenetic analysis of the 16S ribosomal DNA sequences of aphid endosymbionts reveals that they are a monophyletic group with a phylogeny completely concordant with that of their hosts, implying long-term cospeciation. Here we show that rates of base substitution are similar in the 16S ribosomal DNA of different endosymbiont lineages. In addition, we calibrate these rates by assigning age estimates for ancestral aphid hosts to the corresponding endosymbionts. The resulting rate estimates (1-2% per 50 Ma) are among the most reliable available for prokaryotes. They are very near values previously conjectured by using more tenuous assumptions for dating divergence events in eubacteria. Rates calibrated using dates inferred from fossil aphids imply that Asian and American species of the aphid tribe Melaphidina diverged by the early Eocene; this result confirms an earlier hypothesis based on biogeographic evidence. Based on these rate estimates, the minimum age of this endosymbiotic association and the age of aphids as a whole is estimated at 160-280 Ma.