Melanin synthesis in the myxomycete Physarum polycephalum occurs during sporulation but not during spherule formation. Melanin-like pigment was extracted from spores. An almost identical substance of polyphenols was extracted from spherules and characterized by its ultraviolet and infrared absorbance spectra. Polyphenol oxidase activity in spherules was very low and showed only one weak isoenzyme band in isoelectric focusing polyacrylamide gels. A much higher activity, and an increasing number of isoenzymes, were detected in sporulating cultures after illumination during the differentiation process. The addition of melanin precursors resulted in the synthesis of brownish-yellow spherules, probably containing dopachrome, whereas the addition of polyphenol oxidase inhibitors resulted in yellow sporangia. The results indicate that melanin synthesis is probably only a stage in maturation but not an essential part of the morphogenetic process itself.