delta 9-Tetrahydrocannabinol (THC) exposure inhibits numerous immunologic functions of macrophages. The ability of THC-exposed macrophages to provide costimulatory signals to helper T cell hybridomas was investigated by induction of interleukin-2 secretion by T cells in response to immobilized monoclonal anti-CD3 antibody. Exogenous interleukin-1 did not deliver a costimulatory signal to these T cells, suggesting that macrophage costimulatory activity was mediated through cell surface molecules. Modulation of the T cell responses by THC depended on the source of costimulation. THC did not suppress costimulatory activity provided by peritoneal macrophages or immobilized fibronectin. THC at low concentrations markedly diminished the costimulatory activity of a macrophage hybridoma to activate one T cell but not another. Inhibition of costimulation by THC inversely correlated with the loss of activity caused by paraformaldehyde fixation of macrophages. THC at 10(-8) M significantly decreased expression of costimulatory heat-stable antigen, which is resistant to fixation, on the macrophage hybridoma. However, expression of costimulatory B7-1 and B7-2 molecules, which are sensitive to fixation, was not affected by THC. Therefore, THC selectively suppresses a fixation-resistant costimulatory signal to helper T cells in part by diminishing expression of heat-stable antigen.