Growth of Escherichia coli K12 cultivated in minimal medium was strongly inhibited by 2 mM-cyanate. This inhibition could be specifically reversed by arginine. Citrulline (but not ornithine, N-alpha-acetylornithine or N-acetylglutamate) could also restore a normal growth rate. Since growth inhibition by cyanate was followed by an accumulation of ornithine within the cell it was concluded that cyanate specifically inhibits the formation of citrulline from ornithine. The effect of cyanate on the growth of defined strains was consistent with a specific inhibition of carbamoylphosphate synthase. A kinetic study of carbamoylphosphate synthase and ornithine carbamoyltransferase in vitro supported this conclusion. Since carbamoylphosphate is probably the only source of endogenous cyanate it is postulated that carbamoylphosphate synthase activity can be regulated by cyanate resulting from the dissociation of carbamoylphosphate in metabolic circumstances leading to its overproduction.