Three new genes (jadW(1), jadW(2) and jadW(3)) were isolated from a region of the Streptomyces venezuelae ISP5230 chromosome at the left-hand end of the jad cluster for jadomycin B (JdB) biosynthesis. The deduced amino acid sequence of jadW(1) showed strong similarity to gene products associated in several streptomycetes with gamma-butyrolactone autoregulators controlling morphological differentiation and secondary metabolism. Examination of JadW(1) for conserved domains detected a repeat sequence characteristic of proteins in the AfsA regulatory family. Insertional inactivation of jadW(1) reduced the growth rate of S. venezuelae cultures in aerated liquid media containing complex nitrogen sources and altered growth morphology in minimal medium. It also affected sporulation on agar media. Cultures of jadW(1)-disrupted mutants grown under conditions supporting biosynthesis of JdB or chloramphenicol by the wild-type strain failed to produce either of the antibiotics. Complementing the disrupted strain by transformation with pJV435, containing a cloned copy of the gene, improved sporulation and restored antibiotic biosynthesis in transformants to titres close to those of the wild-type similarly transformed with pJV435 as a control. The results are consistent with a role for jadW(1) in regulating morphological and metabolic differentiation. Further sequence analysis of jadR(2), which functions with jadR(1) in stress-induced activation of JdB biosynthesis, indicated that this gene encodes a gamma-butyrolactone receptor homologue. The growth-rate-sensitive phenotype of the jadW(1)-disrupted mutant, and the proximity of jadW(1) to jadR(2) indicate that this region of the jad gene cluster contains a regulatory mechanism incorporating gamma-butyrolactone signalling and sensitivity to environmental stress.