Streptmycetes are widely distributed in nature and are the main producers of biologically active secondary metabolites including many antibiotics. They are also salient in their morphology as prokaryotes forming substrate mycelium, aerial mycelium and spores. In Streptomyces, some congenial autoregulators which control cytodifferentiation and secondary metabolite production are already known. They have a common structural feature, 2,3-disubstituted butanolide skeleton1~4) and are active at nanogram concentrations. We have recently reported the purification and structural elucidation of virginiamycin-inducing factors which were isolated from the culture broth of Streptomyces virginiae. We have isolated three of these factors and found that all of them have the butanolide skeleton. We named them virginiae butanolides (VB's) A (1), B (2) and C (3)4) (Fig. 1). In this report we describe the isolation and structures of two new virginiae butanolides, named VB's D and E, from the culture broth of S. virginiae.