The biosynthetic pathway of 3-amino-5-hydroxybenzoic acid (AHBA) formation was studied with cell-free extracts from the rifamycin B producer, Amycolatopsis mediterranei S699, and the ansatrienin A producer, Streptomyces collinus Tu1892. Phosphoenolpyruvate (PEP) plus erythrose 4-phosphate (E4P) gave AHBA in low but nevertheless significant (6%) yield. 3,4-Dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP) was converted efficiently into AHBA (45%), as were 5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ, 41%) and 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS, 95%). On the other hand, the normal shikimate pathway intermediate, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) did not give rise to AHBA under these conditions. AminoDAHP (9%) was produced by : incubation of [C-14]PEP and E4P, but not of [C-14]DAHP, with the cell-free extracts. The results demonstrate the operation of a new variant of the shikimate pathway in the formation of the mC(7)N units of ansamycin, and presumably also mitomycin, antibiotics which leads from PEP, E4P, and a nitrogen source directly to aminoDAHP and then via aminoDHQ and aminoDHS to AHBA.