Rifabutin, a naphthalenic ansamycin, is a specific inhibitor of bacterial DNA‐directed RNA polymerase and inhibits in vitro the replication of HIV‐1. The synthesis of rifabutinol by PtO2–NaBH4 reduction of rifabutin in ethanol is described. Full assignments in CDCl3 are presented of the 1H and 13C NMR resonance signals using two‐dimensional homonuclear and heteronuclear correlation techniques and selective proton decoupling methods. The three‐dimensional structure of the ansa‐chain of rifabutinol was calculated from NOE contacts and vicinal coupling constants, using a simulated annealing protocol starting from random coordinates. Measurements of the 13C T1 relaxation times of rifabutin in solution are consistent with a rigid ansa‐chain. Copyright © 2000 John Wiley & Sons, Ltd.