Mechanism of transport of the alkylating agent [14C]melphalan was investigated in L5178Y lymphoblasts in vitro. A time course of melphalan uptake was approximately linear for 5 to 10 min and thereafter entered a plateau region. Evidence that unidirectional influx of melphalan is carrier mediated was that uptake obeyed simple Michaelis-Menten kinetics, that it demonstrated chemical specificity, and that the cell/medium distribution ratio of drug decreased with increasing extracellular drug concentration. The kinetic parameters for melphalan transport consisted of a Km (mean +/- S.E.) of 1.53 +/- 0.18 X 10(-4) M and a transport capacity (Vmax) of 3.48 +/- 0.31 X 10(-17) mole/min/cell. Findings suggesting that transport was at least in part energy dependent and not simply a passive process were that drug uptake was temperature sensitive and sodium dependent. Analysis of cell sap constituents indicated the presence of intact drug within the cell. The percentage of radioactivity (mean +/- S.D.) found in the cell sap fraction was 95.8 +/- 2.2% of total cell activity, and 92.6 +/- 4.1% of this was trichloroacetic acid soluble. Thin-layer chromatography of the cell sap fraction and medium each revealed that the majority of radioactivity migrated as a single peak with an RF value identical with that obtained for free drug. The alkylating potential of intact drug complicated interpretation of the finding of apparent uphill transport against a concentration gradient. This observation, together with the relatively low cell-medium ratio (mean +/- S.D.) of 3.07 +/- 1.07, favors the concept that melphalan transport occurs by a facilitated diffusion process, although an active transport system has not been entirely excluded. The relative insensitivity of melphalan uptake to a wide range of metabolic inhibitors also suggests that transport is by a facilitated diffusion mechanism rather than an active process. Other alkylating agents and several amino acids including the L and D isomers of phenylalanine did not inhibit melphalan transport; thus a native substrate was not identified for the melphalan carrier and transport was by a mechanism separate from that of other alkylating agents.