The organic acid probenecid has been shown to interfere with the active extrusion of methotrexate (MTX) from L1210 tumor cells in vitro leading to enhanced MTX accumulation and increased formation of MTX polyglutamate derivatives. In the presence of serum albumin (4 g/100 ml), to which probenecid is bound, the inhibition by probenecid of [3H]MTX efflux from the Ehrlich ascites tumor cell was reduced markedly. While half-maximal inhibition of MTX efflux occurred with 0.12 mM probenecid in the absence of albumin, 1.45 mM probenecid was required in its presence. The presence of albumin also modified the probenecid-induced elevation of steady-state MTX levels in the tumor cell. Maximal elevation of cellular MTX levels occurred with 0.5 mM probenecid in the absence of albumin, and 3 mM probenecid in its presence. Serum albumin further reversed the effects of probenecid on MTX influx. While probenecid inhibited influx of 1 microM [3H]MTX in the absence of albumin (half-maximal inhibition at approximately 1 mM probenecid), probenecid stimulated MTX influx in its presence (half-maximal effect at 0.5 to 1 mM). Equilibrium dialysis studies demonstrated that probenecid displaced MTX from albumin, increasing the effective free concentration of MTX in the incubation medium, and hence the rate of MTX influx. Therefore, probenecid may enhance the accumulation of MTX in the tumor cells by increasing the level of free (as opposed to albumin bound) MTX in the extracellular medium as well as by direct inhibition of MTX efflux. These observations may provide an additional explanation for probenecid enhancement of the therapeutic efficacy of MTX in tumor bearing mice and highlight the importance of assessing drug-protein interactions in an in vitro experimental model.