The 27, 000g supernatant from crude extracts of the dinoflagellate Gonyaulax can be fractionated into two components having luciferase activity that differ both in molecular weight (35, 000 and 150, 000) and in pH activity profile. The smaller component has activity over a broad range from pH 5 to 9, while the larger one is active only in the acid region. This clarifies the previous ambiguity in the literature regarding the optimum pH for the assay of luciferase.