Cryptococcus neoformans, the causative agent of cryptococcosis, produces large amounts of mannitol in culture and in infected mammalian hosts. Although there is considerable indirect evidence that mannitol synthesis may be required for wild-type stress tolerance and virulence in C. neoformans, this hypothesis has not been tested directly. It has been proposed that mannitol-1-phosphate dehydrogenase (MPD) is required for fungal mannitol synthesis, but no MPD-deficient fungal mutants or cDNAs or genes encoding fungal MPDs have been described. Therefore, C. neoformans was purified from a 148 kDa homotetramer of 36 kDa subunits that catalysed the reaction mannitol1-phosphate+NAD--><--fructose 6-phosphate+NADH. Partial peptide sequences were used to isolate the corresponding cDNA and gene, and the deduced MPD protein was found to be homologous to the zinc-containing long-chain alcohol/polyol dehydrogenases. Lysates of Saccharomyces cerevisiae transformed with the cDNA of interest (but not vector-transformed controls) contained MPD catalytic activity. Lastly, Northern analyses demonstrated MPD mRNA in glucose- and mannitol-grown C. neoformans cells. Thus, MPD has been purified and characterized from C. neoformans, and the corresponding cDNA and gene (MPD1) cloned and sequenced. Availability of C. neoformans MPD1 should permit direct testing of the hypotheses that (i) MPD is required for mannitol biosynthesis and (ii) the ability to synthesize mannitol is essential for wild-type stress tolerance and virulence.