This study was planned on the assumptions that different high-voltage activated calcium channels and/or the ability of mitochondria to take up Ca(2+) could be responsible for different cytosolic Ca(2+) concentrations ([Ca(2+)](c)) and catecholamine release responses in adrenal chromaffin cells of bovine and mouse species. Short K(+) pulses (2-5 s, 70 mM K(+)) increased [Ca(2+)](c) to a peak of about 1 microM; however, in bovine cells the decline was slower than in mouse cells. Secretory responses were faster in mouse but were otherwise quantitatively similar. Upon longer K(+) applications (1 min), elevations of [Ca(2+)](c) and secretion were prolonged in bovine cells; in contrast [Ca(2+)](c) in mouse cells declined three-fold faster and failed to sustain a continued secretion. Confocal [Ca(2+)](c) imaging following a 50-ms depolarizing pulse showed a similar Ca(2+) entry, but a rate of [Ca(2+)](c) increase and a maximum peak significantly higher in bovine cells; the rate of dissipation of the Ca(2+) wave was faster in the mouse. The mitochondrial protonophore CCCP (2 microm) halved the K(+)-evoked [Ca(2+)](c) and secretory signals in mouse cells, but had little affect on bovine responses. We conclude that the relative densities of L (15% in bovine and 50% in mouse) and P/Q Ca(2+) channels (50% in bovine and 15% in mouse) do not contribute to the observed differences; rather, the different intracellular distribution of Ca(2+), which is strongly influenced by mitochondria, is responsible for a more sustained secretory response in bovine, and for a faster and more transient secretory response in mouse chromaffin cells. It seems that mitochondria near the plasmalemma sequester Ca(2+) more rapidly and efficiently in the mouse than in the bovine chromaffin cell.