Pantothenic acid (vitamin B5) is the substrate for the biosynthesis of coenzyme A, an essential cofactor in energy metabolism. Because mammals obtain vitamin B5 from their diet, the biosynthesis of pantothenic acid in pathogenic bacteria has been identified as a drug target. Ketopantoate reductase (KPR) catalyzes the NADPH-dependent reduction of ketopantoate (KP) to pantoate, the essential precursor of pantothenic acid (Figure 1). Previous structural and kinetic studies have focused on E. coli KPR, a monomeric enzyme with globular N- and C-terminal domains. Unlike the E. coli enzyme, S. aureus KPR forms a stable, dimeric complex that is highly conserved among several deeply divergent KPRs. Steady state analysis of the S. aureus enzyme also displays positive cooperativity with respect to cofactor. We have shown that the cooperativity is best explained by a random addition mechanism with a kinetically preferred path. Thus, the mechanism of S. aureus KPR is distinct from previously described members of the family of 2-hydroxyacid dehydrogenases. The identification of new drug targets like KPR in S. aureus is important given the increase in multi-drug resistant Staph infections.