The transport mechanisms of 5-aminolevulinic acid methyl ester (5-ALA-ME) have been studied in a human adenocarcinoma cell line (WiDr) by means of ¹⁴[C]-labeled 5-ALA-ME. The transport was found to be partly Na dependent, while the extracellular Cl⁻ concentration did not affect the uptake. The transport of 5-ALA-ME into WiDr cells was dependent on the incubation temperature and was found to be completely blocked by the inhibitors of energy metabolism, 2-deoxyglucose and sodium azide. WiDr cells were treated with 10 mM of 14 different amino acids and the substrate specificity of the 5-ALA-ME transporter(s) was analyzed by treating the cells with 23 μM or 1 mM ¹⁴[C]-labeled 5-ALA-ME. The transport of 5-ALA-ME was found to be inhibited to the highest extent, i.e. about 60%, by the nonpolar amino acids l-alanine, l-methionine, l-tryptophan and glycine. The uptake of 5-ALA-ME followed an exponential decay with increasing concentration of glycine, reaching a maximum inhibition of uptake of 5-ALA-ME of 55%. Sarcosine, a specific inhibitor of system Gly, did not significantly inhibit 5-ALA-ME transport. In contrast to transport of 5-ALA, 5-ALA-ME does not seem to be taken up by system BETA transporters. In conclusion, the cellular uptake of 5-ALA-ME into WiDr cells seems to be due to active transport mechanisms, involving transporters of nonpolar amino acids.