Tumor necrosis factor-a (TNF-a) is a cytokine that induces apoptosis in various cell systems by binding to the TNF receptor (TNFR). To study TNF-a-induced apoptosis, we isolated and characterized a novel TNF-a-resistant variant, U937/ TNF clone UA, from human monocytic leukemia U937 cells. The UA cells resist apoptosis induced by TNF-a and anti-Fas antibody but not by anticancer drugs, such as VP-16 and Ara-C. Somatic cell hybridization between U937 and UA showed that apoptosis resistance to TNF-a in UA was genetically recessive. The hybridization analysis also showed that UA and another recessive mutant clone, UC, belong to different complementation groups in TNF-a-induced apoptosis signaling. In UA cells, TNF-a-induced disruption of mitochondrial membrane potential and CPP32 activation were abrogated. Expression of TNFR, Fas, and Bcl-2 family proteins was not changed in UA cells. These results suggest that the apoptosis resistant UA cells could have a functional defect in apoptosis signaling from the TNFR to mitochondria and interleukin-1b converting enzyme (ICE) family protease activation. UA cells could be used to study signaling linkage between cell death-inducing receptor and mitochondria.