Evidence from our laboratory indicates that microtubules are involved in the differentiation of the dimorphic, pathogenic fungus Histoplasma capsulatum; therefore, we cloned the tubulin genes from a virulent strain of the organism. We report that the H. capsulatum genome contains a single alpha (TUB1) and a single beta (TUB2) tubulin gene rather than the more typical multigene family which is common in even the simplest eukaryotes. Sequence data from these genes reveal a high degree of nucleotide and protein sequence conservation relative to tubulins from other species. The coding regions of TUB1 and TUB2 contain five and eight intervening sequences, respectively. Field inversion gel electrophoresis of H. capsulatum chromosome-sized DNA fragments indicates that the TUB1 and TUB2 genes are unlinked. Potential regulatory elements common to both genes have been identified in the 5' promoter regions. These elements may direct the coordinate expression of TUB1 and TUB2 during differentiation. The cloning and characterization of alpha and beta tubulin genes from H. capsulatum provides the first description of gene structure in this widely distributed pathogenic fungus. Isolation of the tubulin genes will facilitate future studies of tubulin gene expression during the dimorphic phase transitions and clarify the role of microtubules in the differentiation process.