A controversy in the field of bioenergetics has been whether mitochondria are capable of sequestering enough Ca2+ from cytosolic Ca2+ pulses to raise their intramitochondrial free Ca2+ level ([Ca2+]m). This is significant because an increase in [Ca2+]m has been linked to an increase in cellular metabolic rate through various mechanisms. To resolve this question, we exposed isolated liver mitochondria to physiological type pulses of Ca2+ produced using a pulse-generating system (Sparagna, G. C., Gunter, K. K., and Gunter, T. E. (1994) Anal. Biochem. 219, 96-103). We then measured the resulting mitochondrial Ca2+ uptake. The uniporter was previously thought to be the only specific Ca2+ uptake mechanism in mitochondria. Our studies have uncovered an additional uptake mechanism, the rapid mode of uptake or RaM, which functions at the beginning of each pulse and allows mitochondria to sequester a considerable amount of Ca2+ from short pulses. We have shown that the RaM is reset by decreasing the [Ca2+] between pulses for a very short time, making this uptake mode ideally suited for Ca2+ sequestration from Ca2+ pulse sequences. With rapid Ca2+ uptake occurring at the beginning of each pulse, liver mitochondria may be able to sequester sufficient Ca2+ from a short sequence of pulses to activate the cellular metabolic rate.