1. The mitochondrial total calcium content ([Ca]mt) was studied with electron probe microanalysis (EPMA) in isolated guinea-pig ventricular myocytes in order to answer the question of whether electrical stimulation increases [Ca]mt in subsarcolemmal and central mitochondria to a different extent. 2. In unstimulated myocytes subsarcolemmal [Ca]mt was (mean +/- s.e.m.) 535 +/- 229 micromol (kg dry weight (DW))-1 and central [Ca]mt was 513 +/- 162 micromol (kg DW)-1. These values do not differ and correspond to approximately 180 micromol calcium per litre of mitochondria or 180 microM. 3. Contractions were potentiated to an optimum by stimulation with trains of 12 paired stimuli. After potentiation with 12 paired action potentials, cells were shock-frozen 120 ms after the start of the first action potential of the 13th pair. Subsarcolemmal [Ca]mt was 1.3 +/- 0.4 mmol (kg DW)-1 (433 microM) and central [Ca]mt was 227 +/- 104 micromol (kg DW)-1 (76 microM). The difference was significant. 4. After potentiation with 12 paired voltage-clamp pulses, cells were shock-frozen 120 ms after the start of the first pulse of the 13th pair. Subsarcolemmal [Ca]mt was 2.2 +/- 1.0 mmol (kg DW)-1 (733 microM) and central [Ca]mt was 630 +/- 180 micromol (kg DW)-1 (210 microM). After removal of extracellular K+, five paired voltage-clamp pulses increased subsarcolemmal [Ca]mt to 2.1 +/- 0.8 mmol (kg DW)-1 (700 microM), which was significantly higher than the central [Ca]mt of 389 +/- 88 micromol (kg DW) -1 or 130 microM. 5. In unstimulated cells, [Na] and [K] in subsarcolemmal and central mitochondria were not different. In potentiated myocytes, subsarcolemmal [Na]mt was 236 +/- 20 mmol (kg DW)-1 or 79 mM, which is significantly higher than the central [Na]mt of 50 +/- 5 mmol (kg DW)-1 or 16 mM. 6. The differences in [Ca]mt and [Na]mt are attributed to subsarcolemmal cytosolic microdomains of elevated [Ca2+] and [Na+] generated during contractile potentiation by transmembrane Ca2+ and Na+ fluxes.