Studies are presented further characterizing a facilitative system transporting methotrexate (MTX) polyglutamates into lysosomes derived from S180 cells. Initial influx of [3H]MTX + G1 (MTX with 1 additional glutamyl residue) exhibited a slightly alkaline pH optimum (pH 7.7) and was moderately temperature-dependent (Q10 27-37 degrees C = 3.1 +/- 0.1). An analysis of the kinetics of intralysosomal accumulation of [3H]MTX + G1 showed saturation kinetics for initial influx, but linear kinetics for the steady-state level of exchangeable [3H]MTX + G1 at different external concentrations of [3H]MTX + G1. In addition, the system exhibited substantial directional asymmetry with respect to the interaction with MTX + G1 during influx and efflux. Accelerated homo- and heteroexchange diffusion was demonstrated for influx of [3H]MTX + G1, while decelerated homoexchange diffusion was demonstrated for efflux of [3H]MTX + G1 following trans-positioning of MTX + G1 or glutamyl-gamma-glutamate in the opposite compartment. These observations were consistent with a single mobile carrier system mediating influx and efflux of this polyglutamate. Based upon an analysis of competitive interactions with [3H] MTX + G1, this system displayed specificity for MTX-gamma-glutamates, folyl-gamma-polyglutamates, alpha- or gamma-glutamyl peptides and heteropeptides bearing a C-terminal gamma-glutamate but not for MTX or glutamate, themselves. Among polyglutamates, gamma-glutamyl chain length was not a significant factor for transport except in the case of MTX polyglutamates. Overall, our results appear to delineate in the lysosomal membrane a simple mobile carrier system with broad specificity for folyl- or non-folyl-bearing peptides responsible for the transport of MTX polyglutamates.