Abstract Conditions are reported which stabilize pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) activity in extracts of Bacillus subtilis. These conditions include high protein concentrations and the presence of KCl and phosphoenolpyruvate. A partially purified extract was not activated by fructose diphosphate or adenosine monophosphate and not inhibited by adenosine triphosphate. Cells harvested during exponential growth on different carbon sources yielded similar specific activities of pyruvate kinase, indicating the constitutive nature of the enzyme. Nevertheless, the specific activity declined at the end of growth on nutrient sporulation medium.