Evolutionary information derived from the large number of available protein sequences and structures could powerfully guide both analysis and prediction of protein-protein interfaces. To test the relevance of this information, we assess the conservation of residues at protein-protein interfaces compared with other residues on the protein surface. Six homodimer families are analyzed: alkaline phosphatase, enolase, glutathione S-transferase, copper-zinc superoxide dismutase, Streptomyces subtilisin inhibitor, and triose phosphate isomerase. For each family, random simulation is used to calculate the probability (P value) that the level of conservation observed at the interface occurred by chance. The results show that interface conservation is higher than expected by chance and usually statistically significant at the 5% level or better. The effect on the P values of using different definitions of the interface and of excluding active site residues is discussed.