The rate of the non-enzymatic hydrolysis of creatine ethyl ester (CEE) was studied at 37 degrees C over the pH range of 1.6-7.0 using (1)H NMR. The ester can be present in solution in three forms: the unprotonated form (CEE), the monoprotonated form (HCEE(+)), and the diprotonated form (H(2)CEE(2+)). The values of pK(a1) and pK(a2) of H(2)CEE(2+) were found to be 2.30 and 5.25, respectively. The rate law is found to be Rate=-dCCEE/dt=k++[H2CEE2+][OH-]+k+[HCEE+][OH-]+k0[CEE][OH-] where the rate constants k(++), k(+), and k(0) are (3.9+/-0.2)x10(6)L mol(-1)s(-1), (3.3+/-0.5)x10(4)L mol(-1)s(-1), and (4.9+/-0.3)x10(4)L mol(-1)s(-1), respectively. Calculations performed at the density functional theory level support the hypothesis that the similarity in the values of k(+) and k(0) results from intramolecular hydrogen bonding that plays a crucial role. This study indicates that the half-life of CEE in blood is on the order of one minute, suggesting that CEE may hydrolyze too quickly to reach muscle cells in its ester form.