Genes for two structurally and functionally different dihydroorotate dehydrogenases (DHODHs, EC 1.3.99.11), catalyzing the fourth step of pyrimidine biosynthesis, have been previously found in yeast Saccharomyces kluyveri. One is closely related to the Schizosaccharomyces pombe mitochondrial family 2 enzymes, which use quinones as direct and oxygen as the final electron acceptor. The other one resembles the Saccharomyces cerevisiae cytosolic family 1A fumarate-utilizing DHODH. The DHODHs from S. kluyveri, Sch. pombe and S. cerevisiae, were expressed in Escherichia coli and compared for their biochemical properties and interaction with inhibitors. Benzoates as pyrimidine ring analogs were shown to be selective inhibitors of cytosolic DHODs. This unique property of Saccharomyces DHODHs could appoint DHODH as a species-specific target for novel anti-fungal therapeutics.