Protein-tyrosine kinases play pivotal roles in cell signal transduction. We have isolated a cDNA clone encoding a novel human intracytoplasmic tyrosine kinase, termed matk (megakaryocyte-associated tyrosine kinase). Expression of matk mRNA was predominantly found in cells of megakaryocytic lineage. The matk cDNA clone encodes a polypeptide of 527 amino acids and has closest sequence similarity to the csk tyrosine kinase. Sequence comparisons also indicate that matk contains src homology region 2 and 3 domains but lacks the NH2-terminal myristylation signal, the negative regulatory tyrosine (Tyr-527), and the autophosphorylation site (Tyr-416) corresponding to those found in src. Antibodies raised against the NH2 terminus of matk immunoprecipitated a 60-kDa protein from the CMK human megakaryocyte cell line. Expression of matk mRNA was up-regulated in megakaryocytic cells induced to differentiate by the phorbol ester. Based on its restriction in expression and its modulation during in vitro differentiation, it is likely that matk participates in signal transduction during megakaryocytopoiesis.