gamma-Butyrolactones play an important role in the regulation of antibiotic production and differentiation in Streptomyces. However the biosynthetic pathway for these small molecules has not yet been determined, and in vitro synthesis has not been reported. The function of the AfsA family of proteins, originally proposed to catalyse gamma-butyrolactone synthesis, has been in debate. To clarify the function of the AfsA family, and to understand the synthesis of the gamma-butyrolactones, we performed in silico analysis of this protein family. AfsA proteins consist of two divergent domains, each of which has similarity to the fatty acid synthesis enzymes FabA and FabZ. The two predicted active sites in ScbA, which is the AfsA orthologue found in Streptomyces coelicolor, were mutated, and gamma-butyrolactone biosynthesis was abolished in all four constructed mutants, strongly suggesting that ScbA has enzymic activity.