Electron probe microanalysis (EPMA) of a variety of rapidly frozen nonmuscle (e.g., liver and retinal rods) and muscle cell systems indicates that the endoplasmic reticulum (ER) [in muscle, the sarcoplasmic reticulum (SR)] is the major intracellular store of Ca. In vascular smooth muscle, Ca stored in the SR can be released and recycled, and it is sufficient to activate maximal contractions even in those smooth muscles in which the volume of the SR is relatively small. The Ca content of mitochondria in situ in vascular smooth and striated muscles, in liver, and in retinal rods is low, indicating that mitochondria do not function as physiological regulators of cytoplasmic Ca2+ in any of the muscle or nonmuscle cells critically examined with EPMA. Mitochondria themselves may be regulated metabolically by small fluctuations in matrix free Ca2+. Massive accumulation of mitochondrial Ca occurs under pathological conditions, when mitochondria are exposed to abnormally high free Ca2+. In frog skeletal muscle, the return of Ca to the SR is characterized by two processes: a fast one (25% of the Ca released) associated with relaxation due to pumping by the SR, and a slow process (0.4/s) that occurs after relaxation and appears to be rate-limited by the removal of Ca from parvalbumin. Illumination in retinal rods causes no detectable change in the low endogenous Ca content of the outer segment.