Ca2+ loading and retention were examined in isolated skeletal muscle triads and terminal cisternae to determine 1) whether excessive loading altered the response of triads to depolarization-induced Ca2+ release and 2) whether these vesicles were similar in their ability to load and retain Ca2+. A mixture of triads and terminal cisternae was loaded with variable amounts of Ca2+ and then subjected to maximal depolarization. Ca2+ release was monitored by changes in extravesicular fura 2 fluorescence using 340/380-nm excitation and 510-nm emission wavelengths. The amount of Ca2+ released from triads due to maximal depolarization increases with increasing Ca2+ loads until a maximal response is obtained, indicating triad saturation. At pH 7, triadic vesicles preferentially loaded and retained Ca2+ at low Ca2+ loads, but, with increasing loads, nontriadic vesicles began to retain Ca2+. At pH 6.5, which should close all open uncoupled ryanodine receptors, triadic and isolated terminal cisternae vesicles loaded and retained Ca2+ in a similar manner. A population of triads, which have some uncoupled ryanodine receptors, did not retain their loaded Ca2+ at pH 7.0 but did retain Ca2+ at pH 6.5; this resulted in a doubling of the amount of Ca2+ released on maximal depolarization after loading at pH 6.5.