The permeability of isolated sarcoplasmic reticulum (SR) vesicles to monovalent cations was studied using a stopped-flow fluorescence quenching technique that permits the measurement of ion fluxes on a millisecond time scale. Approximately 70% of the SR vesicles carry a cation conductance pathway mediating fluxes of Tl+, K+, Na+, and Li+, but not of choline. Both K+ and Na+ equilibrate faster than the 3-ms dead time of the apparatus and Li+ equilibrates in approximately 50 ms. These cation fluxes are reduced by a bis-guanidinium blocker of the SR K+ channel previously studied in planar bilayers. The remaining 30% of the vesicles are permeable to these cations on a time scale of seconds. We conclude that the SR K+ channel is present in a major fraction of vesicles and that its properties in the native membrane are similar to those found in planar bilayers. Moreover, the ion fluxes in fractionated SR vesicles suggest that the channels are distributed along the entire surface of the SR membrane, but in higher concentration in vesicles derived from the terminal cisternae region. From the measured rates of K+ movement, we calculate a conductance on the order of 10(-1) S/cm2 for the SR membrane in situ, which implies that this membrane cannot develop a potential of more than a few millivolts under physiological conditions.