The new naturally occurring erythromycin G (4), formally derived from erythromycin B by hydroxylation of the C-16 methyl group, and 3-O-mycarosylerythronolide B (5), an erythromycin biosynthetic intermediate previously obtained only from microorganisms blocked in erythromycin biosynthesis, were isolated from a concentrate of mother liquors derived from a culture of Saccharopolyspora erythraea. The structure of erythromycin G was defined by spectroscopic data and X-ray crystallographic analysis. Theoretical calculation of 4 has been performed at MM2 level, and the low-energy conformations have been compared with X-ray data: both theoretical and experimental approaches give similar three-dimensional shapes. Antibacterial activity of 4 against both gram-positive and gram-negative organisms has been evaluated. A simple method for the isolation of large amounts of erythromycins B (2) and D is provided as well.