We have identified and characterized PTK1, a novel human non-receptor protein kinase. Partial PTK1 cDNA was initially isolated by RT-PCR from mRNA of normal human melanocytes. Northern blot hybridization detected a 3.8-kb PTK1 mRNA in all tissues and cell types studied. Nucleotide sequence analysis of a full-length PTK1 cDNA showed it to encode an 847-amino acid polypeptide with an amino-terminal SH3 domain, a kinase catalytic domain, a leucine zipper-like domain, and a carboxyl proline-rich domain. The PTK1 kinase domain contains motifs generally considered diagnostic of serine/threonine kinases but also contains amino acids highly conserved among the tyrosine kinases. This suggests that PTK1 is a serine/threonine kinase, but one very closely related to the tyrosine kinase superfamily. When normal human melanocytes were exposed to antisense PTK1 oligonucleotide, cell growth was inhibited, whereas a corresponding sense oligonucleotide had no inhibitory effect. These data indicate that PTK1 plays an important role in the proliferation of normal human melanocytes.