The level of arginine:glycine amidinotransferase in liver of the developing chick embryo is partially suppressed following injection of arginine into the yolk, and the level can be completely suppressed following injection of guanidinoacetate or creatine (Walker, J.B. (1963), Proc. Soc. Exp. Biol. Med. 112, 245; Walker, J.B., and Wang, S.-H. (1964), Biochim, Biophys. Acta 81, 435). In this investigation structural requirements for the physiological suppressor were examined by testing certain analogues of creatine and its biosynthetic precursors for their ability to suppress liver amidinotransferase levels in developing chick embryos and growing chicks. The creatine analogues, N-acetimidoylsarcosine and 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine), were found to suppress liver amidinotransferase levels of both developing embryos and growing chicks. Compounds ineffective as suppressors included: the arginine analogue, N5-acetimidoylornithine; the guanidinoacetate analogue, N-acetimidoylglycine; and the creatine analogue, 1-carboxymethyl-2-iminohexahydropyrimidine. Our findings suggest that (i) arginine and guanidinoacetate must be converted to creatine before serving as a suppressor, and (ii) creatine, not phosphocreatine, is most closely related to the physiological suppressor of amidinotransferase.