Pyk1 (pyruvate kinase 1) from Saccharomyces cerevisiae was characterized as a substrate for PKA (protein kinase A) from bovine heart and yeast. By designing Pyk1 synthetic peptides containing potential PKA sequence targets (Ser22, Thr94 and Thr478) we determined that the peptide S22 was a substrate for PKA in vitro, with a K(sp)* (specificity constant) 10-fold and 3-fold higher than Kemptide for bovine heart and yeast PKA respectively. In vitro phosphorylation of the Pyk1 S22A mutant protein was decreased by as much as 90% when compared with wild-type Pyk1 and the Pyk1 T94A mutant. The K(sp)* values for Pyk1 and Pyk1 T94A were the same, indicating that both proteins are phosphorylated at the same site by PKA. Two-dimensional PAGE of Pyk1 and Pyk1 S22A indicates that in vivo the S22A mutation prevented the formation of one of the Pyk1 isoforms. We conclude that in yeast the major PKA phosphorylation site of Pyk1 is Ser22. Phosphorylation of Ser22 leads to a Pyk1 enzyme that is more active in the absence of FBP (fructose 1,6-bisphosphate). The specificity of yeast and mammalian PKA towards the S22 peptide and towards whole Pyk1 protein was measured and compared. The K(sp)* for the S22 peptide is higher than that for Pyk1, indicating that the peptide modelled on Pyk1 is a much better substrate than Pyk1, regardless of which tissue was used as the source of PKA. However, the K(m) of Pyk1 protein is lower than that of the better substrate, the S22 peptide, indicating that ground-state substrate binding is not the major determinant of substrate specificity for PKA.