Evaluating the functional relevance of naturally occurring gene variants usually requires experimental testing or is even impossible because of the lack of appropriate functional assays. Here we have analyzed whether comparative sequence data from orthologs are suitable to predict the functional relevance of mutations in a model protein, a G-protein-coupled receptor for ADP (P2Y(12)). The functional effect of every possible substitution at each amino acid position within a portion of P2Y(12) (1254 mutants) was individually determined. Sequence analysis of >70 P2Y(12) vertebrate orthologs revealed that this amino acid variability ensuring proper receptor function in vivo highly correlates (>90%) with the in vitro experimental data. Therefore, ortholog sequence data are helpful to predict the functional relevance of individual positions and mutations for P2Y(12). It is likely that similar conclusions may be extended for other GPCRs and conserved proteins as well.