In the present report, studies are described examining the issue of methotrexate (MTX) polyglutamate retentiveness in cultured L1210 cells. Measurements made in intact L1210 cells showed that the rate of egress of [3H]MTX and [3H]MTX+G1 was different depending upon whether [3H]-MTX and its polyglutamates were accumulated intracellularly following biosynthesis during growth (3 hr) in the presence of [3H]MTX or when cells were pulse loaded (5 min) with [3H]MTX or [3H]-MTX+G1 just prior to measurement of egress. In the former case, [3H]MTX egressed with a T1/2 of 15 +/- 2 min, while [3H]MTX+G1 egressed with a T1/2 of 50 +/- 9 min. In pulse loaded cells, both [3H]MTX and [3H]MTX+G1 egressed with a T1/2 of 3.5 +/- 0.5 min. The same rapid egress of [3H]MTX and [3H]MTX+G1 seen following pulse loading was also documented in cells grown for 3 hr in the presence of nonradioactive MTX to normalize conditions with respect to the intracellular accumulation of [3H]MTX polyglutamates seen during exposure of cells to [3H]MTX during growth. In light of these results, MTX, MTX+G1, MTX+G2 and MTX+G4 were examined directly as permeants for the outwardly-directed ATP-dependent pump (Schlemmer SR and Sirotnak FM, J Biol Chem 267: 14746-14752, 1992) mediating most of MTX efflux in intact L1210 cells using inside-out plasma membrane vesicles isolated from these cells. ATP-dependent efflux of [3H]MTX and [3H]MTX+G1 exhibited values for Km of 46-50 microM and values for Vmax of 102-106 pmol/min/mg protein. As competitive inhibitors of [3H]MTX and [3H]MTX+G1 efflux, MTX+G1 and MTX, respectively, exhibited Ki values of 43-47 microM, that is, Km approximately Ki for both permeants. Also, values for Ki of 45-48 microM were obtained with MTX+G2 and MTX+G4 as competitive inhibitors of [3H]MTX efflux. From these results, we conclude that MTX and its polyglutamates are equivalent as copermeants for ATP-dependent efflux through the plasma membrane and that retentiveness of MTX polyglutamates is not determined at this level in these cells.