γ-Butyrolactone bacterial hormones regulate antibiotic production and morphological differentiation in Streptomyces species. One γ-butyrolactone, SCB1, has been previously characterized in Streptomyces coelicolor. Here we report the characterization of two additional γ-butyrolactones, named SCB2 (2-[1′-hydroxyoctyl]-3-hydroxymethylbutanolide) and SCB3 (2-[1′-hydroxy-6′-methyloctyl]-3-hydroxymethylbutanolide), possessing an antibiotic stimulatory activity. To elucidate the specificity determinants of these ligands for the receptor protein, ScbR, 30 chemically synthesized γ-butyrolactone analogs were tested by utilizing the release of ScbR from DNA upon binding to a γ-butyrolactone, which can be detected by kanamycin resistance. The butyrolactone detection method developed here revealed that ScbR shows preference toward a ligand possessing a 7-10 carbon C-2 side chain, a C-1′-β-hydroxyl group, and a C-6′-methyl branch that coincides with SCB3. Moreover, this method was successfully used to screen for potential γ-butyrolactone producers from commercial-antibiotic-producing Streptomyces. © 2009 Elsevier Ltd. All rights reserved.