The red eye pigment of Drosophila melanogaster consists of six complex pteridines known as neodrosopterin, drosopterin, isodrosopterin, fraction e, and aurodrosopterins (2); these pigments are greatly reduced in the purple mutant. Conditions for biosynthesis of these "drosopterins" are described and compared with those for the synthesis of sepiapterin. The enzymes are contained in a soluble, pteridine-free extract obtained between 40 and 60% saturated ammonium sulfate. The results indicate that sepiapterin synthase consists of two enzymes, the first of which provides a precursor for "drosopterin" biosynthesis. The evidence is (1) the purple mutant, low in accumulated sepiapterin and "drosopterins", is known to have approximately 10% of the sepiapterin synthase activity of wild type; (2) unlabeled sepiapterin does not cause isotope dilution of "drosopterin" synthesis; (3) the 600g pellet prepared from a wild-type head homogenate contains "drosopterin" synthesizing activity and no sepiapterin synthase, yet a heat-labile factor in this fraction stimulates sepiapterin synthesis in the 100000g supernatant of wild-type or pr flies; (4) sepiapterin and "drosopterin" syntheses require Mg2+; (5) sepiapterin synthesis is stimulated by NADPH; "drosopterin" synthesis responds to either NADPH or NADH. Although "drosopterins" are complex pteridine-type pigments, we have demonstrated their biosynthesis by soluble enzymes. This allows us to consider investigation into the mechanism by which the amounts of these pigments are regulated.