As part of a broad program on the genetics of Streptomyces coelicolor A3(2), RUDD and HOPWOOD carried out genetic studies on a nondiffusible red mycelial pigment produced by this organism, obtaining a series of red mutants divided into 5 classes based on cosynthesis studies, with additional genetic work reported by FEITELSON and HOPWOOD. In this study, we investigated the chemical nature of the red pigment. The pigment complex from mutants B18 and 2827 was extracted and analyzed by UV, MS, and 1H NMR spectroscopy, and compared with reference samples. The major components were identified as undecylprodigiosin (orange pigment, confirmed by IR and mixed melting point with an authentic sample) and butylcycloheptylprodiginine (pink pigment, supported by spectral data matching reported values). Mutasynthetic experiments using 3-methoxy-5-phenylpyrrole-2-carboxaldehyde (a bipyrrolealdehyde analog) induced red pigment formation in mutants of classes A, B, C, and E, indicating these mutants are blocked in the bipyrrolealdehyde branch of the prodigiosin biosynthesis pathway—representing the first mutasynthetic modification of the bipyrrole moiety of prodigiosin. Additionally, the ability to synthesize monopyrrole moieties and condense them with a suitable bipyrrolealdehyde analog was found to be more widespread in Streptomycetes. The results identify HOPWOOD's "red pigment" as a mixture predominantly of undecylprodigiosin and butylcycloheptylprodiginine.