The structure of the fluorescent derivative formed in the method of Okayama et al. (Clin Chem 1990; 36:1494-7) for determining delta-aminolevulinic acid (ALA) concentrations was reinvestigated after esterification. The molecular ion peak at m/z 303.1473 corresponded to the molecular formula of C17H21NO4 (calcd 303.1470). The infrared spectrum showed the presence of carbonyl and carboxyl groups. This compound contained two acetyl groups, two methyl groups, and one methoxycarbonylethyl group, as revealed by 1H and 13C nuclear magnetic resonance and 13C-1H shift-correlated spectroscopy. Experiments with correlation spectroscopy via long-range coupling indicated that the main skeleton is 3H-pyrrolizine. The relative arrangement of functional groups was determined by means of nuclear Overhauser effect difference experiments. We were led to the conclusion that the methyl ester of the derivative is 2,6-diacetyl-1,5-dimethyl-7-(2-methoxycarbonylethyl)-3H-pyrrolizine. This structure was unequivocally confirmed by x-ray analysis; therefore, the structure of the derivative itself is 2,6-diacetyl-1,5-dimethyl-7-(2-carboxyethyl)-3H-pyrrolizine.