Both the denaturation, as followed by UV absorbance and fluorescence changes, and inactivation of creatine kinase in quanidine solutions were first order reactions. In 3 M guanidine, at 30.degree. C, the inactivation rate constant was 5.9 sec-1 and the denaturation rate constant 1.9 sec-1. At lower guanidine concentrations, the inactivation rate constants were only little affected whereas the denaturation rate constants decreased markedly, being of the order of 0.04 in 1 M and 0.004 in 0.5 M guanidine. The kinetics of the inactivation reaction in 0.5 M guanidine was in agreement with a combination of 2 first order reactions. The enzyme lost activity first by a fast reaction with a rate constant only slightly lower than the rate constant in 3 M guanidine, very little change in either UV absorbance or in fluorescence was observed, but, in sharp contrast, the enzyme lost considerable activity by a fast reaction and this was followed by a slower reaction of inactivation. Even after prolonged denaturation in 0.5 and 0.3 M guanidine, residual activities of 3.4% and 30% remained respectively. The above results suggest a very fragile active site although dissociation of dimer and reversible guanidine inhibition may also contribute to the initial rapid inactivation. The multiphasic courses of inactivation at lower guanidine concentrations seem to suggest the presence of partly active intermediates during denaturation.