Mitochondrial calcium in rat brain cortex frozen in situ and measured with electron probe X-ray microanalysis was 1.5 mmol Ca/kg dry weight. Cellular, extramitochondrial Ca was 6.4mmol/kg dry weight and cytoplasmic Na+ was 73mM. These results do not support the role of mitochondria regulating cytoplasmic Ca2+, but are compatible with the regulation of mitochondrial enzymes by matrix free Ca2+.