In our studies of metabolic control mechanisms in skeletal muscle from rhesus fetus we have determined the tissue levels of the metabolic intermediates and cofactors of the glycolytic pathway and have calculated the mass-action ratios for each reaction. Skeletal muscle from rhesus fetuses (Macaca mulatta), 90-155 days of gestational age, and from adult rhesus monkeys was used in these experiments. The apparent equilibrium constants for hexokinase and phosphofructokinase (PFK) in these tissues were over 1,000 times larger than the mass-action ratios at all ages studied; the corresponding values for pyruvate kinase were more than 800 times different. The data suggest that these three enzymes are rate-limiting for fetal skeletal muscle as early as 54% of gestation. The next step was to study some of the numerous factors that modify these non-equilibrium reactions. Increasing the ATP concentration had a marked effect on the PFK activity of both fetal and adult muscle, first increasing and then inhibiting enzyme activity. At maximum PFK activity, the amount of fructose-6-PO4(F6P) phosphorylated per mg of protein was 2-3 times greater in the two fetal than in the adult series. At a concentration of 0.3 mM, citrate decreased PFK activity of the 100-day fetal muscle; a further decrease occurred at 1.2 mM citrate. At a citrate level of 0.3 mM. the addition of inorganic phosphate (Pi) or cyclic AMP returned PFK activity to the uninhibited levels (pH 7.0). Relief of ATP inhibition of F6P phosphorylation with Pi and cyclic AMP was also observed at pH 7.0 in extracts of 100-day fetal skeletal muscle.