Two inhibitors of the biosynthesis of aspergillin, the black spore pigment of Aspergillus niger, have been investigated. 2,4-Dithiopyrimidine exerted its inhibitory effect by intracellularly chelating cupric ion required for normal pigmentation. Dimethylsulfoxide prevented the synthesis of certain phenolic precursors of the native pigment. Partial purification and characterization of pigments from mature cultures revealed the presence of at least three components: (i) a high-molecular-weight (-20,000) native pigment fraction in untreated mold cultures, (ii) a lower-molecular-weight (-5,000) melanin pigment found in both types of inhibited cultures, and (iii) a low-molecular-weight (368) green pigment found only in the 2,4-dithiopyrimidine-inhibited cultures and proposed to be a pentacyclic quinonoid derivative. A pathway for aspergillin biosynthesis is suggested based on these results.