1. Intracellular Ca2+ clearance mechanisms were studied in rat adrenal chromaffin cells, by measuring slow tail currents through small-conductance Ca(2+)-activated K+ channels and using indo-1 photometry following depolarization-induced Ca2+ loading. 2. Following several-hundred millisecond depolarizations, [Ca2+]i decayed in three phases. An initial fast decay was followed by a long-lasting, low plateau, then [Ca2+]i returned to the resting level slowly. 3. Replacement of external Na+ moderately slowed [Ca2+]i decay, indicating a contribution of plasma membrane Na(+)-Ca2+ exchange. 4. Raising external pH or application of extracellular Eosin of La3+ prolonged slow tail currents, indicating a contribution of plasma membrane Ca(2+)-ATPase to Ca2+ clearance. 5. Ca(2+)-induced Ca2+ release from caffeine-sensitive stores occurred during depolarization. 6. Inhibition of endoplasmic reticulum Ca(2+)-ATPase had little effect on Ca2+ clearance. 7. Slow tail currents and [Ca2+]i decay following 0.2 - 2 s depolarizations were much prolonged by mitochondrial inhibition with carbonyl cyanide m-chlorophenylhydrazone (CCCP) or Ruthenium Red, which abolished the initial rapid decay and plateau of [Ca2+]i. 8. In conclusion, mitochondrial Ca2+ uptake plays a major role in Ca2+ clearance by rapidly and reversibly sequestering Ca2+ during depolarization-evoked Ca2+ loads.