The Donnan potential in sarcoplasmic reticulum vesicles prepared from rabbit skeletal muscle was measured by using a fluorescent cyanine dye, diS-C3-(5) (3,3'-dipropylthiadicarbocyanine iodide). The Donnan potential is caused by the existence of negative fixed charges inside the vesicles. On average, sarcoplasmic reticulum vesicles have internal negative fixed charges of 420 nmol/mg protein at pH 6.8. The fixed charges are mainly due to low affinity Ca2+ binding proteins. The Donnan potential in sarcoplasmic reticulum vesicles was about -72 mV in 5 mM K-MES buffer (pH 6.8). When Ca2+ was accumulated into sarcoplasmic reticulum vesicles with ATP, the concentration of negative fixed charges decreased from 140 mM to 95 mM due to Ca2+ binding to the negative fixed charges, and sarcoplasmic reticulum vesicles were depolarized by about 10 mV. It was found that there are two types of sarcoplasmic reticulum vesicles, one type (60%) having many negative fixed charges inside the vesicles and the other (40%) having few fixed charges.