Virginiae butanolide A (VB A) (3) is one of the virginiamycin-inducing factors from Streptomyces virginiae and has a unique 2,3-disubstituted butanolide skeleton which is common to other signal molecules in Streptomyces. The biosynthesis of 3 in Streptomyces antibioticus, a high producer of 3, was studied by experiments with labeled precursors. I3C and *H NMR results as well as CI-MS analyses of dibenzoate samples indicated that the probable biosynthetic pathway to 3 involved coupling between a 8-keto acid derivative and a C3 unit from glycerol, such as dihydroxyacetone or a derivative.Signal molecules which regulate secondary metabolite production or cytodifferentiation of Streptomyces have been known for nearly 20 years. The first one was A factor (1) found by Khokhlov et al., which induces formation of streptomycin, aerial mycelium, and spores in Streptomyces griseus.' After the discovery of A factor, Grlfe et al. isolated factor 1 (2) from the Recently we found a strain of Streptomyces antibioticus which produces several milligrams of VB A per liter of culture broth,9 and this finding has made it possible to elucidate the biosynthesis of 3 by feeding experiments with Wlabeled precursors. We have detected. (8) (a) Kim, H. S.; Tada, H.; Nihira, T.; Yamada, Y. J. Anribiot. 1990, 43,692-706. (b) Kim, H. S.; Nihira, T.; Tada, H.; Yamada, Y. Ibid. 1989, 42, 769-778. (c) Miyake, K.; Horinouchi, S.; Yoshida, M.; Chiba, N.; Mori, K.; Nogawa, N.; Morikawa, N.; Beppu, T.Abstract: Pyrrolosine, a C-nucleoside recently isolated from Streptomyces albus, was assigned structure 1 by Ikegami and ceworkers largely on the basis of single-crystal X-ray diffraction. This is also the structure previously assigned to the synthetic C-nucleoside, 9-deazainosine. Because the physical and biological properties of the two compounds differ, Ikegami and co-workers suggested that the structure of 9-deazainosine should be reinvestigated. However, it is shown in this paper that the structure they proposed for pyrrolosine is incorrect. Furthermore, on the basis of the reported physical properties, it is shown that pyrrolosine is actually the known furo[3,2-d]pyrimidine C-nucleoside adenosine analogue 2. This conclusion, which is more consistent