The activities of K+-dependent pyruvate kinase and malate dehydrogenase in cell-free extracts from Saccharomyces cerevisiae X2180-1B and R. rubra NCYC 797 were examined in the presence of Cs+ and other monovalent cations (Li+, Na+, K+, Rb+, NH4+) in order to assess their ability to substitute for K+ in activation of these enzymes and therefore their suitability as probes for studying the importance and function of K+. Varying the monovalent cation concentration (up to 100 mM) in S. cerevisiae did not inhibit activity of either of the enzymes assayed. In comparison, NADPH-dependent malate dehydrogenase from R. rubra was inhibited approximately 90±1% in the presence of K+, Cs+, Na+, Rb+ and Li+ at a concentration of 100 mM. In contrast, NADH-dependent malate dehydrogenase from R. rubra showed constant enzyme activity irrespective of the monovalent cation present indicating that NADH-dependent malate dehydrogenase may be of low monovalent cation specificity in both yeasts or possesses no binding sites for monovalent cations apart from K+. Similarly, Cs+ and the other monovalent cations tested did not affect the activity of pyruvate kinase from S. cerevisiae. In contrast, pyruvate kinase from R. rubra was less inhibited by cations with ionic radii approximating more closely to that of K+ (133 pm). NH4+ (143 pm) and Rb+ (147 pm) could partially substitute for K+ in enzyme activation, whereas smaller ions such as Na+ (97 pm) or Li+ (78 pm) or larger ions such as Cs+ (166 pm) inhibited activity. Kinetic analysis of pyruvate kinase from R. rubra revealed an increased Vmax of 14·4 × 10-2 μmol lactate produced min-1 (mg protein)-1 at high [K+] (50-200 mM) compared to a Vmax of 7·5 × 10-2 μmol lactate produced min-1 (mg protein)-1 at low [K+] (1-25 mM) which indicated the presence of additional K+-binding sites on the enzyme; Km values of 42·5 and 4·4 mM were obtained for high (50-200 mM) and low (1-25 mM) K+ concentration ranges respectively. Inhibition of K+ activation of pyruvate kinase by Cs+ was examined over the K+ concentration range 1-25 mM K+ and Cs+ concentrations of 25-100 mM. Kinetic analysis revealed that Cs+ inhibition was predominantly uncompetitive over these concentration ranges indicating that the Cs+ combines with the enzyme-activator (K+) complex and not with the free enzyme to form an inactive enzymatic structure. It is concluded that Cs+-sensitive K+-dependent enzymes, such as pyruvate kinase from R. rubra, may represent an intracellular site for toxicity of inessential monovalent cations.