Probenecid has been shown to delay the plasma clearance of methotrexate in the rat and to reduce both hepatic and renal excretion of methotrexate in this animal model. In order to probe the mechanism by which probenecid alters hepatic excretion of the antifolate, studies assessed the effects of probenecid on transport, accumulation, distribution, and release of methotrexate in the rat hepatocyte in suspension. Probenecid was found to effectively inhibit methotrexate influx with a Ki of approximately 100 microM. Inhibition of methotrexate influx was accompanied by a reduction in methotrexate accumulation; with 200 microM probenecid, the levels of exchangeable and nonexchangeable intracellular methotrexate were reduced by 43.4 +/- 2.4 (S.E.) and 41.8 +/- 7.7%, respectively. As a consequence of reduced accumulation of the methotrexate substrate, the formation of cellular polyglutamate derivatives of methotrexate was likewise reduced. Concentrations of probenecid which inhibited methotrexate influx and accumulation by 70 to 80% did not markedly alter methotrexate efflux under conditions where efflux was effected by a washout procedure or by the presence of inducing agents, such as N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate or alpha-agonists. These studies suggest that the inhibition of hepatic methotrexate secretion by probenecid in vivo is likely to be a consequence of interference with hepatic uptake of the antifolate rather than an interaction of probenecid and methotrexate at a hepatic "secretory" site.