We examined the hypothesis that both misoprostol (miso), a prostaglandin E(1) (PGE(1)) analog, and nonsteroidal anti-inflammatory drugs (NSAIDs) have significant and largely inhibitory effects on T-cell activation and, consequently, influence T-cell function. Studies were done using a macrophage-independent system for T-cell activation by mitogenic combinations of immobilized anti-T-cell monoclonal antibodies (mabs), including combinations of anti-CD3 with anti-CD4 or anti-CD6. The results indicate that misoprostol, like other prostaglandins, can inhibit T-cell proliferation and the expression of mRNAs for cytokines critical in T-cell growth and immunoregulation. Somewhat higher concentrations of misoprostol are required than of PGE(1) to achieve comparable effects. However, even very low doses of misoprostol (0.01 &mgr;g ml(minus sign1)) may sometimes enhance T-cell activation. The effects on cytokine mRNA are not uniform; for a given dose and agent, one cytokine mRNA may be enhanced while another is unchanged or diminished. Whether a response is increased or decreased depends on several factors, including the dose of misoprostol or other PG tested, the strength of the response on which the agent is acting, and the level of preexisting activation or cytokine gene expression. Similar findings can be seen with treatment in vitro with NSAIDs, including sodium salicylate. When PGs (including misoprostol) and NSAIDs are combined, effects are often additive and sometimes appear synergistic, but paradoxical results can also occur, whereby each agent separately may be modestly inhibitory, but together they are stimulatory. The in vitro response of T cells to both PGs and NSAIDs (either separately or together) is, thus, highly complex. These data support the notion that these agents may have significant effects on the regulation of T-cell activation and function.