We have previously demonstrated circadian variation of pyruvate kinase (PK) activity. PK is a key enzyme of glycolysis, whose activity has been shown to be regulated at the level of enzyme concentration and catalytic activity. To investigate the mechanism of regulation and the components of the circadian rhythm of PK activity, Michaelis-Menten kinetic values were determined. C57BL/6J male mice that had been standardized for over 2 weeks to 12 hr light followed by 12 hr dark (lights on at 0600 hr CST), were killed at 3-hr intervals (six mice/circadian stage) over a 24-hr span. Vmax values, which are a measure of enzyme concentration, were found to be characterized by circadian variation (P = 0.01 by ANOVA, but P = 0.38 by cosinor analysis). The time of highest observed Vmax occurred at the early dark and continued through the entire dark and early light spans. An abrupt decrease in Vmax (-40%) was observed at the end of the light span. PK catalytic activity is regulated by covalent modification (phosphorylation/dephosphorylation) of the enzyme, and the phosphorylated, inactive form of the enzyme has a high Km for its major substrate (1.41 mM phosphoenol pyruvate; PEP). Km values were found to be characterized by a circadian rhythm (P less than 0.001 by cosinor analysis) whose acrophase (1117 hr) was at the beginning of the light cycle (high Km indicates low catalytic activity at subsaturating PEP concentrations). These results indicate that the circadian rhythm for PK activity is composed of at least two components. Enzyme concentration is high during the dark and early light phases but decreases at the end of the light phase. Concentration recovers rapidly within 3 hr after the lights are turned off. Catalytic activity is maximal at a time just after enzyme concentration becomes maximal in the dark phase. Maximal efficiency of the enzyme is thus achieved at the time when animals are eating and glycolysis is operating at high levels.