A human gene encoding a protein that specifically binds to the intracellular domain of the 75 kDa type-2 tumour necrosis factor (TNF) receptor (TNFR-2IC) has been identified using the yeast-based two-hybrid system. The N-terminal half of the TNF receptor-associated protein (TRAP) contains RING finger and zinc finger motifs often found in DNA-binding proteins including transcription factors. The 2.4 kb TRAP mRNA was barely detectable, if present at all, in lung, and variably expressed in heart, liver, placenta, brain, skeletal muscle, kidney and the pancreas; interestingly, the TRAP was more highly expressed in transformed cell lines than in normal tissues. This observation may be consistent with a role for this TRAP in promoting or regulating cellular proliferation. After in vitro transcription/translation and 35S labelling the TRAP was precipitated using a fusion protein consisting of glutathione S-transferase and the intracellular domain of TNFR-2 (TNFR-2IC), which showed that the two proteins directly interact in a mammalian cell-free system and also that identification of the TRAP was not an artifact of the two-hybrid system. By using truncated TNFR-2ICs for in vitro precipitation of 35S-TRAP, it was shown that the C-terminal half of the TNFR-2IC contains the domain necessary for interaction with TRAP. The TRAP identified in the present study shares considerable homology with, and may be the human homologue of, a mouse protein, TNF receptor-associated factor 2 (TRAF2), that binds mouse TNFR-2.