p19 is a highly conserved 19-kDa cytosolic protein that undergoes phosphorylation in mammalian cells upon activation of several distinct signal transduction pathways. Its expression is widespread but developmentally regulated. To determine the in vivo phosphorylation site(s) of p19, the protein was purified from bovine brain and resolved into the unphosphorylated form (p19) and a mixture of the two predominant phospho-forms (pp19). Proteolytic fragments of p19 and pp19 were examined by liquid chromatography/mass spectrometry (LC/MS). We detected ion masses corresponding to fragments spanning the entire amino acid sequence as deduced from the cDNA except for those predicted to contain an unmodified amino terminus. Instead, the digests revealed ions corresponding to peptides lacking the initiator methionine and containing an N-acetylated alanine at the amino terminus. The analysis of pp19, but not that of p19, revealed two sets of ions representing peptides whose m/z values differed by 80 atomic mass units, the incremental mass of a phosphate residue. These putative phosphate-bearing peptides were sensitive to alkaline phosphatase treatment. Using combined trypsin and V8 protease digestions, the phosphorylation sites were mapped to Ser-25 and Ser-38, in the peptides Leu-Ile-Leu-Ser*-Pro-Arg and Phe-Pro-Leu-Ser*-Pro-Pro-Lys, respectively. Interestingly, both phosphoserines are in a very similar sequence context, suggesting that a single proline-directed serine protein kinase, possibly p34cdc2, is responsible for phosphorylation of both sites in vivo.