Carbonic anhydrase (CA) activity was determined in a homogenate of frog skeletal muscle by measuring the kinetics of CO2 hydration in a pH stopped-flow apparatus. The results suggest that frog skeletal muscle contains a high-activity CA with properties similar to those of the isoenzyme CA II found in white skeletal muscle tissue of the rabbit. In an attempt to assess the functional significance of CA in skeletal muscle, the maximal isometric force of frog gastrocnemius muscle was measured in response to direct or indirect (ischiadic nerve) single-pulse electrical stimulation before (control) and after exposing the muscle to various concentrations of the specific carbonic anhydrase inhibitors, ethoxzolamide, acetazolamide, and methazolamide. In the range of ethoxzolamide concentration between 10(-9) and 10(-6) M, maximal isometric force with indirect supramaximal stimulation declined progressively with inhibitor concentration to less than 10% of the control value. Acetazolamide and methazolamide were less effective in that concentrations of above 10(-4) M were necessary to inhibit maximum isometric force by 50%. Even at the highest ethoxzolamide concentration used (10(-6) M), no effect was observed either on the amplitude of the compound nerve action potential or on the conduction velocity of group I fibres in the ischiadic nerve, suggesting that ethoxzolamide did not affect the mechanisms responsible for spike generation or conduction in the motor fibres. With direct supramaximal stimulation of the gastrocnemius muscle, no effects on maximal isometric force were observed of CA inhibition by any of the inhibitors used. The results suggest that CA acts on the neuromuscular transmission. The exact site and mechanism of action are unknown.