Carbonic anhydrase (CA) accelerates contractile function, particularly in fast-twitch skeletal muscles. Since the extraocular muscles are considered to be amongst the fastest skeletal muscles in mammals, this study tested two hypotheses: (1) CA is expressed at higher levels in rat extraocular muscles than in extensor digitorum longus (EDL, a fast limb muscle), and (2) inhibition of CA activity increases twitch duration and force in the extraocular muscles to a greater extent than in EDL. By real-time quantitative PCR we determined that the expression of CA3 isoform, typically high in skeletal muscles, is significantly depressed in extraocular muscles. Message levels for the CA2 and CA4 isoforms were higher in the extraocular muscles, while CA5 expression was equivalent in both muscles. Strong CA activity was demonstrated by histochemistry in frozen EDL muscle sections, in particular along the sarcolemma and in capillaries. By contrast, extraocular muscle had very low sarcolemmal or cytosolic CA activity. CA inhibition with 6-ethoxyzolamide (ETZ) reversibly increased twitch duration and force in EDL muscle bundles. In the extraocular muscles, ETZ did not alter twitch kinetics. Based on these results, we reject our initial hypotheses and conclude that CA does not influence the fast contractile kinetics characteristic of the extraocular muscles.