Differentiation of human T cells into T helper (Th)1 and Th2 cells is vital for the development of cell-mediated and humoral immunity, respectively. However, the precise mechanism responsible for the Th1 cell differentiation is not fully clarified. We have studied the expression and function of Txk, a member of the Tec family of nonreceptor tyrosine kinases. We found that Txk expression is restricted to Th1/Th0 cells with IFN-gamma producing potential. Txk transfection of Jurkat T cells resulted in a several-fold increase of IFN-gamma mRNA expression and protein production; interleukin (IL)-2 and IL-4 production were unaffected. Antisense oligodeoxynucleotide of Txk specifically inhibited IFN-gamma production of normal peripheral blood lymphocytes, antigen-specific Th1 clones, and Th0 clones; IL-2 and IL-4 production by the T cells was unaffected. Txk cotransfection led to the enhanced luciferase activity of plasmid (p)IFN-gamma promoter/enhancer (pIFN-gamma[-538])-luciferase-transfected Jurkat cells upon mitogen activation. Txk transfection did not affect IL-2 and IL-4 promoter activities. Thus, Txk specifically upregulates IFN-gamma gene transcription. In fact, Txk translocated from cytoplasm into nuclei upon activation and transfection with a mutant Txk expression plasmid that lacked a nuclear localization signal sequence did not enhance IFN-gamma production by the cells, indicating that nuclear localization of Txk is obligatory for the enhanced IFN-gamma production. In addition, IL-12 treatment of peripheral blood CD4(+) T cells enhanced the Txk expression, whereas IL-4 treatment completely inhibited it. These results indicate that Txk expression is intimately associated with development of Th1/Th0 cells and is significantly involved in the IFN-gamma production by the cells through Th1 cell-specific positive transcriptional regulation of the IFN-gamma gene.