Guanidine-induced alterations in substrate-dependent kinetics of glycine amidinotransferase (GAT) have been investigated in homogenates of rat kidney. Guanidine hydrochloride (GuHCl) induced a mixed type of inhibition by decreasing the maximal velocity (Vmax) and increasing the Michaelis-Menten constant (Km). The finding of a value of Ki smaller than that of Ki' denoted that the inhibition of GAT may be due to decreased E to S affinity rather than to reduction in the active site density of the enzyme.