Cannabichromenic acid synthase was purified to apparent homogeneity by sequential column chromatography including DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The enzyme catalysed the oxidocyclization of cannabigerolic acid and cannabinerolic acid to cannabichromenic acid. The K(m) values for both substrates were in the same order of magnitude although the Vmax value for the former was higher than that for the latter. These results suggested that cannabichromenic acid is predominantly formed from cannabigerolic acid rather than cannabinerolic acid. The enzyme required neither molecular oxygen nor hydrogen peroxide, indicating that the cannabichromenic acid synthase reaction proceeds through direct dehydrogenation without hydroxylation.