Abstract TCR stimulation results in the tyrosine phosphorylation of a number of cellular substrates. We have recently identified a 70-kDa protein tyrosine kinase, ZAP-70, which associates with the human TCR zeta-chain after TCR stimulation. We report here the isolation and sequence of a cDNA clone that encodes murine ZAP-70. Murine and human ZAP-70 share 93% amino acid identity and are homologous to the 72-kDa protein tyrosine kinase Syk. Syk has been implicated in the signal transduction pathways of the B cell membrane Ig and high affinity IgE receptors, Fc epsilon RI. In addition, we examined the tissue distribution of ZAP-70 and Syk in human and murine thymocyte subsets, B cells, and peripheral T cell subsets. ZAP-70 protein is expressed in all major thymocyte populations, with the level of expression being comparable to that found in both CD4+ and CD8+ peripheral T cells. Although Syk protein is also present in all thymocyte subsets, expression of Syk protein is down-regulated threefold to fourfold in peripheral T cells. In contrast to ZAP-70, expression of Syk is 12- to 15-fold higher in peripheral B cells when compared with peripheral T cells. In addition, whereas T cell stimulation results in down-regulation of Lck, no significant change in ZAP-70 or Syk protein is detected. Finally, we provide evidence that both ZAP-70 and Syk can associate with the TCR after TCR stimulation. With the use of a heterologous expression system, we show that, like ZAP-70, Syk is dependent upon a Src-family protein tyrosine kinase for association with the phosphorylated zeta-chain. Thus, the differential expression of these kinases suggests the possibility of different roles for ZAP-70 and Syk in TCR signaling and thymic development.