At least 70% of small cell lung cancers (SCLCs) express the Kit receptor tyrosine kinase and its ligand, stem cell factor (SCF). In an effort to define the signal transduction pathways activated by Kit in SCLC, we focused on Src family kinases and, in particular, Lck, a Src-related tyrosine kinase that is expressed in hemopoietic cells and certain tumors, including SCLC. SCF treatment of the H526 cell line induced a physical association between Kit and Lck that, in vitro, was dependent on phosphorylation of the juxtamembrane domain of Kit. Stimulation of Kit with recombinant SCF resulted in a rapid 3-6-fold increase in the specific activity of Lck, which was similar in magnitude to the activation of Lck resulting from the cross-linking of the T-cell receptor complex of Jurkat cells. Lck activity peaked by 5 min after SCF addition, and the elevated activity persisted for at least 30 min in the presence of SCF, with kinetics similar to the activation of mitogen-activated protein kinase. PP1, an inhibitor of Src family kinases with selectivity for Lck, completely inhibited SCF-mediated growth but had little effect on insulin-like growth factor-I-mediated growth. PP1 antagonized both SCF-mediated proliferation and inhibition of apoptosis. PP1 had no effect on Kit kinase activity but was shown to block total Lck activity by at least 90% by immune complex kinase assay. Low levels of Src, Hck, and Yes were also expressed in the H526 cell line; only Yes showed a consistent increase in specific activity, which was also inhibited by PP1 following SCF treatment. These data demonstrate that, in the H526 SCLC cell line, Lck and, possibly, Yes are downstream of Kit in a signal transduction pathway; the inhibition by PP1 of SCF-mediated proliferation and inhibition of apoptosis suggests that Src family kinases are intermediates in the signaling pathways that regulate these processes.