Bioluminescence in all dinoflagellate species studied to date is produced by the luciferase-catalyzed oxidation of a newly elucidated type of luciferin, hypothesized to have a substituted polypyrrole-type structure. This paper presents the purification and characterization of the luciferin from Pyrocystis lunula along with evidence that it is a polypyrrole-type molecule. Luciferin is extremely labile at low pH, at high salt concentration, and to O2, so, where possible, the purification steps were carried out in the presence of a buffered reducing agent and under argon. Purified luciferin is soluble in water and polar organic solvents. It is yellow (lambda max 245 and 390 nm with a shoulder at 290 nm in neutral or basic aqueous solution) and displays a strong blue fluorescence (lambda max for excitation at 390 nm, for emission at 474 nm) that closely matches the bioluminescence emission spectrum [Bode, V. C., & Hastings, J. W. (1963) Arch. Biochem. Biophys. 103, 488--499]. Autoxidation leads to concomitant decreases in the 390-nm absorbance, 474-nm fluorescence, and biological activity; similar changes occurred with oxidation by K3Fe(CN)6, thus allowing a quantitation of luciferin by titration. Luciferin has a molecular weight between 500 and 600, displays positive Ehrlich and Schlesinger reactions, and yields on acid chromate oxidation fragments apparently resembling substituted maleimides; these data support the proposal that dinoflagellate luciferin contains a substituted polypyrrole of the bile pigment type.