Mitochondrial Ca(2+) activates many processes, from mitochondrial metabolism to opening of the permeability transition pore (PTP) and apoptosis. However, there is considerable controversy regarding the free mitochondrial [Ca(2+)] ([Ca(2+)](M)) levels that can be attained during cell activation or even in mitochondrial preparations. Studies using fluorescent dyes (rhod-2 or similar), have reported that phosphate precipitation precludes [Ca(2+)](M) from increasing above 2-3 microM. Instead, using low-Ca(2+)-affinity aequorin probes, we have measured [Ca(2+)](M) values more than two orders of magnitude higher. We confirm here these values by making a direct in situ calibration of mitochondrial aequorin, and we show that a prolonged increase in [Ca(2+)](M) to levels of 0.5-1mM was actually observed at any phosphate concentration (0-10mM) during continuous perfusion of 3.5-100 microM Ca(2+)-buffers. In spite of this high and maintained (>10 min) [Ca(2+)](M), mitochondria retained functionality and the [Ca(2+)](M) drop induced by a protonophore was fully reversible. In addition, this high [Ca(2+)](M) did not induce PTP opening unless additional activators (phenyl arsine oxide, PAO) were present. PAO induced a rapid, concentration-dependent and irreversible drop in [Ca(2+)](M). In conclusion [Ca(2+)](M) levels of 0.5-1mM can be reached and maintained for prolonged periods (>10 min) in phosphate-containing medium, and massive opening of PTP requires additional pore activators.