We have studied the correlation between the degree of filling of the intracellular Ca2+ stores and the plasma-membrane permeability to Mn2+, a Ca2+ surrogate for plasma-membrane Ca2+ channels, in human neutrophils loaded with fura-2. Refilling of the stores of cells previously depleted of Ca2+ decreased the entry of Mn2+, but the magnitude of this effect depended on the refilling protocol. When refilling was allowed to proceed to steady-state levels by a 3 min incubation with different external Ca2+ concentrations (0.05-1 mM), almost complete inhibition of Mn2+ entry was observed at 40% of maximum refilling. In contrast, when different degrees of store refilling were attained by incubation with 1 mM-Ca2+ for short periods (10-40 s), inhibition of Mn2+ entry was smaller at comparable degrees of refilling. When quick refilling was allowed to proceed up to 40% (about 20 s at 37 degrees C) and then stopped at this level by removal of external Ca2+, the rate of Mn2+ uptake was high just after refilling and then decreased with time within the next few seconds (half-times approximately 7 s at 37 degrees C and approximately 20 s at 25 degrees C). We have proposed previously that the Ca2+ stores, when emptied of Ca2+, may generate a second messenger able to open the plasma-membrane Ca2+ channels by a mechanism involving cytochrome P-450. The results here are consistent with the existence of such a messenger and suggest that it is cleared from the cytoplasm with a half-time of about 7 s at 37 degrees C. In addition, inhibition of Mn2+ entry in cells with empty Ca2+ stores by cytochrome P-450 inhibitors showed a time lag consistent with the clearance kinetics proposed above.