The mshB gene encoding N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis. Disruption of mshB in Mycobacterium smegmatis resulted in decreased production of mycothiol (5-10 % of the parent strain mc(2)155) but did not abolish mycothiol synthesis completely. Complementation of the MshB(-) mutants with the mshB gene resulted in increased mycothiol production towards the exponential and stationary phases of the bacterial growth cycle. These results suggest that another enzyme is capable of mycothiol biosynthesis by providing N-acetylglucosaminylinositol deacetylation activity in the absence of MshB. One of the candidate enzymes capable of carrying out such reactions is the MshB orthologue mycothiol amide hydrolase, MCA. However, epichromosomal expression of mca in the MshB(-) mutants did not restore mycothiol levels to the level of the parent strain. Unlike other mutants, which have little or no detectable levels of mycothiol, the MshB(-) mutant did not exhibit increased resistance to isoniazid. However, the MshB(-) mutant was resistant to ethionamide. Phenotypic analysis of other mutants lacking mycothiol revealed that MshA(-) mutants also exhibit ethionamide resistance but that a MshC(-)mutant was sensitive to ethionamide, suggesting that mycothiol or its early intermediates influence ethionamide activation.