Objective: Our aim was to elucidate the molecular roles of carbonic anhydrases CA IV, CA IX and CA XIV in the transfer of lactic acid across the sarcolemma of the fast extensor digitorum longus (EDL) of the mouse. Methods: We studied lactic acid fluxes by measurements of intracellular pH_i, using muscle fiber bundles and pH microelectrodes. Influx of lactic acid via the lactate-proton cotransporter MCT causes a fall in pH_i over time, whose slope, multiplied by the intracellular buffer factor, gives the rate of lactic acid transport into the cell. We investigated muscle fibers from single-, double- and triple- CA-ko mice. Results: The rate of lactic acid influx is not significantly different in CA IV-ko EDL and WT EDL, but fluxes in both CA XIV- and CA IX-single-ko EDL are ~30% lower than in WT EDL. Lactic acid fluxes in EDL from CA IV-CA XIV- double- and CA IV-CA IX-CA XIV-triple-ko mice were reduced more strongly, by ~50% compared to WT EDL. Thus, lack of all three membrane-bound CAs reduces lactate transport to ½, which we show to be the transport rate in the absence of any functional CA. These electrophysiological results are complemented by immunocytochemical evidence showing that CA IV and CA XIV are exclusively localized in the surface membrane, while CA IX is exclusively localized in the T-tubule. Conclusion: There are two pathways of CA-dependent lactic acid transfer across the sarcolemma: one across the surface membrane requiring CO₂ hydration catalysis by CA IV/CA XIV and lost when the two isozymes are lacking, another one across the T-tubular membrane that requires CA IX. The finding that lack of CA IX causes a marked reduction in lactic acid flux, whilst having hardly an effect when knocked out in addition to CA IV and CA XIV, suggests that the T-tubular pathway requires a cooperation of CA IX with CA IV/CA XIV and vanishes, when either CA IX alone or CA IV plus CA XIV are lacking. We propose a detailed mechanism explaining these findings.