Relaxation studies using the temperature jump have been carried out on pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) isolated from rabbit muscle. Several concentration-independent relaxation effects with associated relaxation times of approx. 1·10−4 sec were observed in the presence of some of the activator divalent metal cations and substrates used. These effects are attributed to isomerizations or conformational changes of the enzyme. The appearance and disappearance of these effects, as observed in both the visible (with a pH indicator) and ultraviolet region, as a function of the temperature range studied 915–36°) indicate that distinct conformational states of the enzyme exist and that the proportion of each state varies with temperature and the liganded state of the protein. Although a large range of enzyme and substrate concentrations were examined relatively few relaxation processes could be detected. This indicates that either a suitable detection system could not be found and/or many of the reactions associated with the enzymatic reaction are too rapid to observe.