The influence of recombinant interferon-gamma (rIFN-gamma) on the development of acetylcholine receptor (AChR) aggregates in cocultures of rat embryonic muscle cells and spinal cord neurons was studied by counting the number of AChR aggregates in relation to cholinergic nerve fibers coming to the muscle fibers. rIFN-gamma caused no decrease in the number of cholinergic nerve fibers, but inhibited the increase in the number of AChR aggregates that occurs early during cocultivation and is an early sign in the development of neuromuscular junctions. rIFN-gamma stimulated release of nitric oxide, but no effects on aggregation of AChRs occurred after exposure to a nitric oxide synthase inhibitor, L-NG-monomethylarginine, or by the addition of nitroprusside, a generator of nitric oxide. No effect was seen on the number of AChR aggregates when the cultures were exposed to rIFN-gamma at later time points of cocultivation, when the increase in number of AChRs had already occurred. These studies indicate that the key immunoregulatory cytokine IFN-gamma can cause alterations in the early process of synapse formation and that these effects are independent of the nitric oxide release caused by the cytokine.