Several members of the Actinomycetales, including the medically important mycobacteria, produce 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amino-2-deoxy-alpha-D- glucop yranoside (trivial name mycothiol) as their principal low-molecular-mass thiol. The pseudo-disaccharide component of mycothiol, 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (alpha-D-GI), was synthesized by ligation of 1-D,L-2,3,4,5, 6-penta-O-acetyl-myo-inositol to 3,4,6-tri-O-acetyl-2-deoxy- 2-(2,4-dinitrophenylamino)-alpha-D-glu- copyranosyl bromide to give, in the first instance, an isomeric mixture of alpha- and beta-linked pseudo-disaccharides. The alpha-coupled D,D and D,L isomers, alpha-D-GI and alpha-L-GI respectively, were purified from the mixture by TLC, followed by removal of the protecting groups. A cell-free extract of Mycobacterium smegmatis catalysed the ligation of cysteine, acetate and alpha-D-GI in the presence of ATP and Mg2+ to form mycothiol, as judged by HPLC. When no acetate was added to the incubation mixture, an additional thiol accumulated. In the presence of [14C]acetate no radiolabel was recovered in this species, but only in mycothiol. The additional thiol was isolated as the bimane derivative, and 1H and 1H-1H COSY NMR spectra confirmed its identity as desacetylmycothiol. A more complete conversion of desacetylmycothiol into mycothiol was achieved in the presence of acetyl-S-CoA. These results indicate that the biosynthesis of mycothiol proceeds by the sequential addition of cysteine and acetate to alpha-D-GI. The inositol moiety appears to be an important determinant of specificity, since alpha-L-GI was poorly utilized.