An afsA homologue (srrX) and three gamma-butyrolactone receptor gene homologues (srrA, srrB and srrC) are coded on the giant linear plasmid pSLA2-L in Streptomyces rochei 7434AN4, a producer of two polyketide antibiotics, lankacidin and lankamycin. Construction of gene disruptants and their phenotypic study revealed that srrX and srrA make a gamma-butyrolactone receptor system in this strain. Addition of a gamma-butyrolactone fraction to an srrX-deficient mutant restored the production of lankacidin and lankamycin, indicating that the SrrX protein is not necessary for this event. In addition to a positive effect on antibiotic production, srrX showed a negative effect on morphological differentiation. The receptor gene srrA reversed both effects of srrX, while the second receptor gene homologue srrC had only a positive function in spore formation. Furthermore, disruption of the third homologue srrB greatly increased the production of lankacidin and lankamycin. Electron microscopic analysis showed that aerial mycelium formation stopped at a different stage in the srrA and srrC mutants. Overall, these results indicated that srrX, srrA, srrB and srrC constitute a complex regulatory system for antibiotic production and morphological differentiation in S. rochei.