5-aminolevulinic acid (5-ALA) and its ester derivatives are used in photodynamic therapy as precursors for the formation of photosensitizers. This study relates to the mechanisms by which 5-ALA is transported into cells. The transport of 5-ALA has been studied in a human adenocarcinoma cell line (WiDr) by means of [¹⁴C]-labeled 5-ALA. The rate of uptake was saturable following Michaelis–Menten kinetics (K_m = 8–10 mM and V_max = 18–20 nmol·(mg protein × h)⁻¹), and Arrhenius plot of the temperature-dependent uptake of 5-ALA was characterized by a single discontinuity at 32°C. The activation energy was 112 kJ·mol⁻¹ in the temperature range 15°–32°C and 26 kJ·mol⁻¹ above 32°C. Transport of 5-ALA was Na and partly Cl⁻-dependent. Stoichiometric analysis revealed a Na :5-ALA coupling ratio of 3:1. With the exception of valine, methionine and threonine, zwitterionic and basic amino acids inhibited the transport of 5-ALA. 5-ALA methyl ester was not an inhibitor of 5-ALA uptake. The transport was most efficiently inhibited, i.e. by 65–75%, by the β-amino acids, β-alanine and taurine and by γ-aminobutyric acid (GABA). Accordingly, 5-ALA, but not 5-ALA methyl ester, was found to inhibit cellular uptake of [³H]-GABA and [¹⁴C]-β-alanine. Protoporphyrin IX (PpIX) accumulation in the presence of 5-ALA (0.3 mM) was attenuated 85% in the presence of 10 mM β-alanine, while PpIX formation in cells treated with 5-ALA methyl ester (0.3 mM) or 5-ALA hexyl ester (4 μM) was not significantly influenced by β-alanine. Thus, 5-ALA, but not 5-ALA esters, is transported by β-amino acid and GABA carriers in this cell line.