Of a number of divalent cations investigated, Zn2+ strongly inhibited lactate production from glucose 6-phosphate in rat muscle cytosol fraction. The I50 value for lactate production influenced by Zn2+ was 10 microM and was increased to 200 microM and 18 microM by the addition of 10mM histidine and 10mM carnosine, respectively. The inhibitory effect of 50 microM Zn2+ on lactate production was completely reversed by the addition of 1.2mM histidine and the apparent Km was found to be 0.34mM. The inhibitory site for Zn2+ was investigated by the estimation of glycolytic intermediates. It occurred at point between fructose 6-phosphate and fructose 1,6-bisphosphate. Purified rat muscle phosphofructokinase was inhibited by Zn2+. The I50 values for Zn2+ were calculated to be 12, 5.5 and 1.5 microM in the presence of 3, 0.3 and 0.06mM fructose 6-phosphate, respectively. The addition of histidine removed the inhibitory effect of Zn2+ on the glycolytic key enzyme. These studies indicate that in rat skeletal muscle the inhibitory effect of Zn2+ on lactate production might result from the inhibition of phosphofructokinase and that histidine could remove the effect.