We recently cloned the cDNA which encodes a novel megakaryocyte-associated tyrosine kinase termed MATK. In this study, we have cloned and characterized the human MATK gene as well as the murine homolog of human MATK cDNA and performed functional studies of its translated product. Comparison of the deduced amino acid sequences of human and murine MATK cDNAs revealed 85% homology, indicating that MATK is highly conserved in mouse and human. The human gene consists of 13 exons interrupted by 12 introns. The genetic units which encode the SH3 and SH2 domains are located on separate exons. The putative ATP binding site (GXGXXG) is localized on exon 7, and the entire catalytic domain is subdivided into seven exons (7-13). Somatic cell hybrid analysis indicated that human MATK gene is located on chromosome 19 while the murine Matk gene is located on chromosome 10. The immediate 5'-flanking region was highly rich in GC sequences, and potential cis-acting elements were identified including several SP1, GATA-1, APRE, and APRE1. Antisense oligonucleotides directed against MATK mRNA sequences significantly inhibited megakaryocyte progenitor proliferation. Functional studies indicated that MATK can phosphorylate the carboxyl-terminal conserved tyrosine of the Src protein. These results support the notion that MATK acts as a regulator of p60c-src in megakaryocytic cells and participates in the pathways regulating growth of cells of this lineage.