Transcription and complementation experiments were carried out to analyze the regulatory hierarchy of two Streptomyces antibiotic regulatory protein (SARP) genes, srrY and srrZ, in the gamma-butyrolactone (GB)-dependent regulatory cascade in Streptomyces rochei 7434AN4. The srrY gene was transcribed in the srrZ mutant, while the srrZ gene was not in the srrY mutant. The SrrY protein was specifically bound to the promoter region of srrZ, where a possible SARP binding sequence was identified 26 bp upstream of the -10 sequence. Deletion of the repeat sequences from this region abolished its SrrY binding activity. In addition, complementation of srrZ restored lankamycin production in the srrY mutant. All of these results confirm that the SARP gene srrY directly regulates expression of the second SARP gene srrZ in a positive manner. This study gave the first confirmation of direct regulation of two SARP genes in the GB-dependent regulatory cascade in Streptomyces.