We have found previously that Txk, a member of the Tec family tyrosine kinases, is involved importantly in T helper 1 (Th1) cytokine production. However, how Txk regulates interferon (IFN)-gamma gene transcription in human T lymphocytes was not fully elucidated. In this study, we identified poly(ADP-ribose) polymerase 1 (PARP1) and elongation factor 1alpha (EF-1alpha) as Txk-associated molecules that bound to the Txk responsive element of the IFN-gamma gene promoter. Txk phosphorylated EF-1alpha and PARP1 formed a complex with them, and bound to the IFN-gamma gene promoter in vitro. In particular, the N terminal region containing the DNA binding domain of PARP1 was important for the trimolecular complex formation involving Txk, EF-1alpha and PARP1. Several mutant Txk which lacked kinase activity were unable to form the trimolecular complex. A PARP1 inhibitor, PJ34, suppressed IFN-gamma but not interleukin (IL)-4 production by normal peripheral blood lymphocytes (PBL). Multi-colour confocal analysis revealed that Txk and EF-1alpha located in the cytoplasm in the resting condition. Upon activation, a complex involving Txk, EF-1alpha and PARP1 was formed and was located in the nucleus. Collectively, Txk in combination with EF-1alpha and PARP1 bound to the IFN-gamma gene promoter, and exerted transcriptional activity on the IFN-gamma gene.