1. The mechanisms governing the return of intracellular calcium (Cai2+) to baseline levels following depolarization-evoked [Ca2+]i rises were investigated in Purkinje cell somata using tight-seal whole-cell recordings and fura-2 microfluorometry, for peak [Ca2+]i ranging from 50 nm to 2 microM. 2. Cai2+ decay was well fitted by a double exponential with time constants of O.6 and 3 s. Both time constants were independent of peak [Ca2+]i but the contribution of the faster component increased with [Ca2+]i. 3. Thapsigargin (10 microM) and cyclopiazonic acid (50 microM) prolonged Cai2+ decay indicating that sarco-endoplasmic reticulum Ca2+ (SERCA) pumps contribute to Purkinje cell Cai2+ clearance. 4. A modest participation in clearance was found for the plasma membrane Ca2+ (PMCA) pumps using 5,6-succinimidyl carboxyeosin (40 microM). 5. The Na(+)-Ca2+ exchanger also contributed to the clearance process, since replacement of extracellular Na+ by Li+ slowed Cai2+ decay. 6. Carbonyl cyanide m-chlorophenylhydrazone (CCCP, 2 microM) and rotenone (10 microM) increased [Ca2+]i and elicited large inward currents at -60 mV. Both effects were also obtained with CCCP in the absence of external Ca2+, suggesting that mitochondrial Ca2+ uptake uncouplers release Ca2+ from intracellular stores and may alter the membrane permeability to Ca2+. These effects were irreversible and impeded tests on the role of mitochondria in Cai2+ clearance. 7. The relative contribution of the clearance systems characterized in this study varied as a function of [Ca2+]i. At 0.5 microM Cai2+, SERCA pumps and the Na(+)-Ca2+ exchanger contribute equally to removal and account for 78% of the process. Only 45% of the removal at 2 microM Cai2+ can be explained by these systems. In this high [Ca2+]i range the major contribution is that of SERCA pumps (21%) and of the Na(+)-Ca2+ exchanger (18%), whereas the contribution of PMCA pumps is only 6%.