Rat heart mitochondria were incubated for 5 min at 30 degrees C and at approx. 40 mg protein.ml-1 and in the presence of 10 microM fura-2/AM. This allowed the entrapment of free fura-2 within the mitochondrial matrix and its use as a probe for Ca2+, but without affecting the apparent viability of the mitochondria. Parallel measurements of the activities of the intramitochondrial Ca2+-sensitive enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, allowed an assessment of their sensitivity to measured free Ca2+ within intact mitochondria incubated under different conditions; the enzymes responded to matrix Ca2+ over the approximate range 0.02-2 microM with half-maximal effects at about 0.3-0.6 microM Ca2+. Effectors of Ca2+-transport across the inner membrane (e.g., Na+, Mg2+, Ruthenium red, spermine) did not appear to affect these ranges, but did bring about expected changes in Ca2+ distribution across this membrane. Significantly, when mitochondria were incubated in the presence of physiological concentrations of both Na+ and Mg2+, and at low extramitochondrial Ca2+ (less than 400 nM), there was a gradient of Ca2+ (in:out) of less than unity; at higher extramitochondrial [Ca2+] (but still within the physiological range) the gradient was greater than unity indicating a highly cooperative nature of transmission of the Ca2+ signal into the matrix under such conditions.