Newly hatched chicks fed a commercial diet containing 1% cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) rapidly accumulate in breast muscle a derivative of cyclocreatine; within a few days concentrations up to 35 mumol of this derivative/g fresh weight of muscle are attained. The following evidence suggests that this derivative is N-phosphorylated cyclocreatine. (a) The derivative is adsorbed by Dowex 1 resin and eluted near phosphocreatine. (b) The derivative present in muscle is converted to free cyclocreatine either by homogenization of muscle in water at room temperature, conditions under which endogenous creatine kinase is active, or by heating a cold acidic muscle extract at 65 degrees for 45 min. (c) The isolated derivative reacts in vitro with the specific reagents crystalline creatine kinase and MgADP to give cyclocreatine. Essentially all of breast muscle cyclocreatine appears to be in the form of P-cyclocreatine, which persists in muscle long after cyclocreatine is removed from the diet. Long term conservation of P-cyclocreatine in muscle is aided by the fact that, unlike P-creatine, P-cyclocreatine is not continuously degraded to an inactive cyclic lactam. It is suggested that the maximal concentrations of P-cyclocreatine2- (and P-creatine2-) attained in sarcoplasm not only affect the phosphorylation potential of muscle cells, but also can account for more than half of the normal inorganic cation concentration of muscle sarcoplasm, and hence play an important role in muscle function. Other chick tissues active in accumulation of cyclocreatine are heart (up to 20 mumol/g fresh weight within 11 days on the diet) and brain (up to 10 mumol/g fresh weight after 30 days on the diet). Addition of 1% creatine to the diet of cyclocreatine-fed chicks does not prevent accumulation of cyclocreatine in muscle. Chicks fed cyclocreatine do not grow as rapidly as those on control diets, but they appear healthy, and mortality is very low when oxytetracycline is added to the drinking water. Cyclocreatine is also taken up by rat muscle, heart, and brain. A sensitive and specific assay for cyclocreatine has been developed. Cyclocreatine reacts with an aged aqueous solution of Na3[Fe(CN)8NH3] under alkaline conditions to give a blue product with a molar absorption coefficient (epsilon) of 4,400 M(-1) cm(¿ at 605 nm. The following compounds give an epsilon605 in this assay of less than 4 M%¿ cm(% N-phosphorylcyclocreatine, creatine, P-creatine, creatinine, 1-carboxymethyl-2-iminohexahydropyrimidine, guanidinoacetate, 3-guanidinopropionate, and arginine. Cyclocreatine does not interfere with the diacetyl-alpha-naphthol assay for creatine.