The specificity of TPK-IIA, a spleen tyrosine protein kinase whose activity is dramatically enhanced by either polylysine or very high ionic strength, has been inspected with the aid of a variety of very short peptide substrates. Unlike free tyrosine, which is not phosphorylated at all, and the dipeptides YA and AY, whose phosphorylation is hardly detectable, the tripeptides AYA, YYY and YYA, tetrapeptide AYAA and pentapeptide AAYAA are readily phosphorylated, the V m a x of AYA being 7- and 15-fold lower vs those of AYAA and AAYAA, respectively. In comparison to AYA, the tripeptides YAA, EYA, GYA and VYV are poorer substrates, while EYH is unaffected by TPK-IIA. The rate of phosphorylation of the pentapeptide EYAA is negligible as compared to AYAA; the hexapeptide EEYAA, however, is a substrate quite comparable to AAYAA, exhibiting a somewhat lower V m a x but also a 4-fold lower K m (3.9 vs 18.6 mM). Taken together, the data would indicate that although TPK-IIA does not exhibit any absolute requirement for a definite consensus sequence it is nevertheless endowed with peptide substrate specificity, mainly resulting from negative determinants that can be variably overcome by additional features of the sequence.