1. Ventricular myocytes isolated from ferret or cat were loaded with the acetoxymethyl ester form of indo-1 (indo-1 AM) such that approximately 75% of cellular indo-1 was mitochondrial. The intramitochondrial indo-1 concentration was 0.5-2 mM. 2. Myocytes were also voltage clamped (membrane capacitance, Cm = 100 pF) and a typical wash-out time constant of cytosolic indo-1 by a patch pipette was found to be approximately 300 s. Depolarizations to +110 mV produced graded and progressive cellular Ca2+ load via Na(+)-Ca2+ exchange. 3. During these relatively slow Ca2+ transients, cell contraction (delta L) paralleled fluorescence ratio signals (R) such that delta L could be used as a bioassay of cytosolic [Ca2+] ([Ca2+]c), where [Ca2+]CL is the inferred signal which is delayed by approximately 200 ms from true [Ca2+]c. 4. In myocytes without Mn2+ quench, the kinetics of the total cellular indo-1 signal, delta R (including cytosolic and mitochondrial components), match delta L during stimulations at low basal [Ca2+]i. However, after progressive Ca2+ loading, delta R kinetics deviate from delta L dramatically. The deviation can be completely blocked by a potent mitochondrial Ca2+ uniport blocker, Ru360. 5. When cytosolic indo-1 is quenched by Mn2+, initial moderate stimulation triggers contractions (delta L), but no change in indo-1 signal, indicating both the absence of cytosolic Ca(2+)-sensitive indo-1 and unchanged mitochondrial [Ca2+] (delta [Ca2+]m). Subsequent stronger stimulation evoked larger delta L and also delta R. The threshold [Ca2+]c for mitochondrial Ca2+ uptake was 300-500 nM, similar to that without Mn2+ quench. 6. At high Ca2+ loads where delta [Ca2+]m is detected, the time course of [Ca2+]m was different from that of [Ca2+]c. Peak [Ca2+]m after stimulation has an approximately 1 s latency with respect to [Ca2+]c, and [Ca2+]m decline is extremely slow. 7. Upon a Ca2+ influx which increased [Ca2+]c by 0.4 microM and [Ca2+]m by 0.2 microM, total mitochondrial Ca2+ uptake was approximately 13 mumol (1 mitochondria)-1. 8. With Mn2+ quench of cytosolic indo-1, there was no mitochondrial uptake of Mn2+ until the point at which mitochondrial Ca2+ uptake became apparent. However, after mitochondrial Ca2+ uptake starts, mitochondria continually take up Mn2+ even during relaxation, when [Ca2+]c is low. 9. It is concluded that mitochondria in intact myocytes do not take up detectable amounts of Ca2+ during individual contractions, unless resting [Ca2+]c exceeds 300-500 nM. At high cell Ca2+ loads and [Ca2+]c, mitochondrial Ca2+ transients occur during the twitch, but with much slower kinetics than those of [Ca2+]c.