Three different mammalian myo-inositol cotransporters are currently known; two are Na+-coupled (SMIT1 and SMIT2) and one is proton-coupled (HMIT). Although their transport stoichiometries have not been directly determined, significant cooperativities in the Na+ activation of SMIT1 and SMIT2 suggest that more than one Na+ ion drives the transport of each myo-inositol. The two techniques used here to determine transport stoichiometry take advantage of the electrogenicity of both SMIT2 and HMIT expressed in Xenopus oocytes. The first method compares the measurement of charge transferred into voltage-clamped oocytes with the simultaneous uptake of radiolabelled substrate. The second approach uses high accuracy volume measurements to determine the transport-dependent osmolyte uptake and compares it to the amount of charge transported. This method was calibrated using a potassium channel (ROMK2) and was validated with the Na+/glucose cotransporter SGLT1, which has a known stoichiometry of 2 : 1. Volume measurements indicated a stoichiometric ratio of 1.78 +/- 0.27 ion per alpha-methyl-glucose (alphaMG) for SGLT1 whereas the radiotracer uptake method indicated 2.14 +/- 0.05. The two methods yielded a SMIT2 stoichiometry measurement of 1.75 +/- 0.30 and 1.82 +/- 0.10, both in agreement with a 2 Na+:1 myo-inositol stoichiometry. For HMIT, the flux ratio was 1.02 +/- 0.04 charge per myo-inositol, but the volumetric method suggested 0.67 +/- 0.05 charge per myo-inositol molecule. This last value is presumed to be an underestimate of the true stoichiometry of one proton for one myo-inositol molecule due to some proton exchange for osmotically active species. This hypothesis was confirmed by using SGLT1 as a proton-driven glucose cotransporter. In conclusion, despite the inherent difficulty in estimating the osmotic effect of a proton influx, the volumetric method was found valuable as it has the unique capacity of detecting unidentified transported substrates.