Kinase of embryonic stem cells (Kos1), a nonreceptor protein tyrosine kinase (NRPTK), was identified and cloned from differentiating murine embryonic stem cells. Kos1 is localized on mouse chromosome 11 that corresponds to human chromosome 17p13. 1 and is homologous to Tnk1, Ack1 and Ack2, making it a new member of the Ack family of NRPTKs. Kos1 is a ubiquitously expressed 47-kDa protein with autotyrosine kinase activity that is developmentally regulated during embryogenesis. Kos1 is also upregulated following IL3 withdrawal from factor-dependent murine NSF/N1. H7 cells that undergo apoptosis, suggesting a role in growth inhibition. Stable overexpression of Kos1 inhibits growth of NIH 3T3 cells, while the kinase-dead Kos1 (CN) promotes cell growth in both liquid culture and soft agar. In addition, forced expression of Kos1 inhibits Ras activity in an indirect mechanism that results in the downregulation of the Ras–Raf1–MAPK growth pathway. Furthermore, overexpression of Kos1 in NCI-H82 lung cancer cells that express oncogenic Ha-Ras (G12V) inhibits cell growth under reduced serum (0.5%) conditions in close association with the upregulation of the Ras inhibitor, Rap1A. Collectively, these data support a negative regulatory role for Kos1 in regulating the Ras–Raf1–MAPK growth pathway by a mechanism that requires its autotyrosine kinase activity.