The Streptomyces virginiae barA gene encodes a specific receptor protein for virginiae butanolide (VB), one of the gamma-butyrolactone autoregulators of Streptomyces species. By homologous recombination, a barA null strain was constructed to clarify the in vivo function of BarA protein in S. virginiae. The Delta barA mutant showed no difference in terms of growth, but lost VB production and produced virginiamycin 7h earlier than the wild-type strain. These results indicated that, phenotypically, BarA protein acts negatively in virginiamycin biosynthesis and positively in VB biosynthesis. Furthermore, Northern (RNA) blot analysis of the Delta barA mutant revealed that transcription of the BarA target gene (barB) was derepressed, confirming that BarA acts as a transcriptional repressor in S. virginiae.