Drosopterins are one of the major groups of pigments present in the eyes of Drosophila Isolation of this red pigments was first reported by Lederer in 1940 and subsequently Pfleiderer proposed the chemical structure which contains both pteridine and pyrimidodiazepine (homopterin) ring systems. The elucidation of the structure had initiated the biochemical studies that include the investigation of enzymes leading to the biosynthesis of this compounds. The enzymes responsible for this pathway are GTP cyclohydrolase I, 6-pyruvoyl-H₄-pterin synthase, and pyrimidodiazepine synthase. Recently, it has been demonstrated that the final step i.e., the conversion from H₂-pterin and acetylhomopterin to drosopterins can be carried out nonenzymatically. The only remaining step in the pathway is therefore the production of H₂-pterin. Since the three-carbon side chain of a 6-substituted pterin must be eliminated during this process, a rapid, simple method has been developed to assay the enzymatic release of the side chain. The "side chain releasing activity" could be measured effectively by filtration of the radioactive reaction products through a Pasteur pipet packed with charcoal and DEAE Sephadex. Using this assay, we were able to purify the enzyme from the extracts of Drosophila. Some of the enzymatic characteristics of this new enzyme have been determined. Although the enzyme preparation is not pure enough to identify the released three-carbon compound and the other pterin product, we found that neither neopterin nor neopterin-P could replace H₂-neopterin triphosphate as substrate. In this report, evidence will also be presented that the enzyme activity is involved in the formation of drosopterin as well as other simpler pteridines such as pterin, xanthopterin, and isoxanthopterin. There are at least 20 genetically well known eye color mutants deficient in the drosopterin pigments. The biochemical studies of the drosopterin pathway with these mutant strains will offer a unique tool to investigate the gene-enzyme relationships in Drosophila.