Dihydroorotate dehydrogenase is a critical enzyme of de novo pyrimidine biosynthesis in prokaryotic and eukaryotic cells. Differences in the primary structure of the enzymes from Gram-positive and -negative bacteria and from mammals indicate significant structural divergence among these enzymes. We have identified a class of small molecules, the thiadiazolidinediones, that inhibit prototypical enzymes from Gram-positive and -negative bacteria, but are inactive against the human enzyme. The most potent compound in our collection functioned as a time-dependent irreversible inactivator of the bacterial enzymes with k(inact)/K(i) values of 48 and 500 M(-1) sec(-1) for the enzymes from Escherichia coli and Enterococcus faecalis, respectively. The data presented here indicate that it is possible to inhibit prokaryotic dihydroorotate dehydrogenases selectively while sparing the mammalian enzyme. Thus, this enzyme may represent a valuable target for the development of novel antibiotic compounds.