A novel protein tyrosine kinase not related to pp60c-src, designated as N-PTK, has recently been found in neonatal rat brain. In the present study, the enzyme was purified further by heparin-Sepharose column chromatography, and identified as a monomer protein with a Mr of 47 K and a pI of 7.0 by two-dimensional gel electrophoresis. The enzyme was found to phosphorylate purified pp60c-src at a tyrosine residue(s). The major phosphorylation site was shown by alpha-chymotryptic peptide mapping to be in the carboxy terminal V8 protease fragment (V2), but to be different from the autophosphorylation site, Tyr-416. The phosphorylation significantly suppressed pp60c-src activity with enolase as a substrate. These findings strongly suggest that N-PTK is a specific kinase that phosphorylates pp60c-src and regulates its function in the cell.