Mycobacterium tuberculosis and related members of the genus Mycobacterium contain a number of inositol-based lipids, such as phosphatidylinositol mannosides, lipomannan and lipoarabinomannan. The synthesis of phosphatidylinositol in M. smegmatis is essential for growth and myo-inositol is a key metabolite for mycobacteria. Little is known about the biosynthesis of inositol in mycobacteria and the only known de novo pathway for myo-inositol biosynthesis involves a two-step process. First, cyclization of glucose 6-phosphate to afford myo-inositol 1-phosphate via inositol-1-phosphate synthase and, secondly, dephosphorylation of myo-inositol 1-phosphate by inositol monophosphatase (IMP) to afford myo-inositol. The following report examines IMP activity in M. smegmatis extracts, with regard to pH dependence, bivalent cation requirement, univalent cation inhibition, regulation by growth and carbon source. We show that IMP activity, which is optimal at the end of the exponential growth phase in Sauton's medium, is Mg(2+)-dependent. Moreover, IMP activity is inhibited by Li(+) and Na(+), with Li(+) also being able to inhibit growth of M. smegmatis in vivo. This study represents a first step in the delineation of myo-inositol biosynthesis in mycobacteria.