Nitric oxide synthase (NOS, EC 1.14.23) catalyzes the oxidation of the guanidino-nitrogen of L-arginine to form nitric oxide and L-citrulline. 2-Iminobiotin was found to be a reversible inhibitor of murine iNOS and rat n-cNOS with Ki values of 21.8 and 37.5 microM, respectively. The urea and thiourea analogs, biotin and thiobiotin, were not inhibitors of NOS, indicating that the guanidino group of 2-iminobiotin is essential for binding. 2-iminobiotin carboxy derivatives were also inhibitors of iNOS which indicates that the free carboxyl group is not required for binding. 2-Iminobiotin is a novel, potent inhibitor of NO biosynthesis and may be a useful reagent in the understanding of binding-site interactions for this important class of enzymes.