We describe a protocol for quantitative labeling of tryptic peptides with oxygen-18. Proteins are first digested in natural water with trypsin, the pH is then lowered to 4.5 and the mixture is dried. Oxygen-18 water is added and two oxygen-18 atoms are incorporated at the peptides' carboxyl termini. Trypsin is finally inactivated by cysteine alkylation under denaturing conditions, which blocks oxygen back-exchange. The general value of this labeling strategy for differential proteomics is illustrated by the analysis and identification of several couples of differently labeled amino terminal peptides isolated from a human platelet proteome by a previously described chromatographic procedure.