The lck gene encodes a membrane-associated protein tyrosine kinase that is expressed specifically in lymphoid cells, especially thymocytes. Structural analysis of the murine and human lck genes previously identified conserved 5' flanking sequences that were proposed to represent transcriptional regulatory elements. Here we demonstrate that a murine lck promoter construct containing these sequences directs the expression of the SV40 T-antigen gene in lymphoid cells. Remarkably, expression of SV40 T-antigen in transgenic animals dramatically disturbs thymic development, resulting in preferential loss of CD4+CD8+ thymocytes. In contrast, immature cells lacking both CD4 and CD8 markers are present in near-normal numbers. Thus SV40 T-antigen expression appears partially to arrest thymopoiesis. Mice bearing the lck-SV40 transgene develop readily explantable thymic tumors at 12-18 weeks of age. Fluorocytometric analyses of lck-SV40 tumor cells reveal that immature thymocytes are frequently immortalized. The lck-SV40 mouse may therefore provide materials for the in vitro investigation of thymocyte differentiation.