5-aminolevulinic acid (ALA) is the committed biological precursor to porphyrins. At supraphysiological concentrations ALA can dimerize to form 3,6-dihydropyrazine-2,5-dipropanoic acid (DHPY), which transfers electrons to XTT in a reaction that does not require metal ions and is specifically inhibited by superoxide dismutase. The formation of DHPY from ALA follows dimerization kinetics with a pK of 7.8+/-0.1. At pH 11.2, DHPY is relatively stable, but when the pH is dropped to 6.0 rapid conversion to 2,5-(beta-carboxyethyl)pyrazine occurs via an intermediate with an absorption maximum of 370 nm. Formation of this intermediate is pH-dependent with a pK of 6.0+/-0.1. These data indicate that ALA dimerizes to produce superoxide from a protonated form of DHPY. The significance of these results with respect to the concentrations of ALA used in photodynamic therapy, and the increased incidence of liver cancer in acute intermittent porphyria, is discussed.