Several studies have postulated a transport of H+ into the sarcoplasmic reticulum (SR) during Ca++ release as well as a transport of H+ out of the SR during Ca++ uptake, occurring to partly compensate the charges transferred in the form of Ca++ . The H+ ions required for these fluxes may be generated from the CO2/HCO3- buffer system. This system has to be catalyzed by carbonic anhydrase (CA) because the half-time of the uncatalyzed reaction is far too slow compared to the fast kinetics of Ca++ fluxes. In order to study, which CA isozyme might be involved in this process, fibers of the M. extensor digitorum longus (EDL) from mouse were examined by double immunostaining using anti- CA/FITC with anti-SERCA/TRITC as well as anti-CA/FITC with anti-RyR/TRITC antibodies. By CLSM, we found that CA XIV is localized in the longitudinal SR, whereas CA IX is expressed in the terminal SR/T-tubular region, and CA IV is present in both regions. Western blots using isolated SR membrane vesicles of wildtype (WT) skeletal muscles demonstrated a 39 kDa, a 43 kDa and a 54 kDa band representing CA IV, CA IX, and CA XIV. EDL fibers of WT and single-CA-knockout (KO) mice were used to investigate isometric single twitches, tetanic force and fatigue. The muscle function of CA-KO fibers did not differ from the function of WT fibers. It is concluded that lack of one CA in the SR membrane seems to be compensated by the other CA isozymes.