Bovine chromaffin cells possess several types of Ca2+ channels, and influx of Ca2+ is known to trigger secretion. However, discrepant information about the relative importance of the individual subtypes in secretion has been reported. We used whole-cell patch-clamp measurements in isolated cells in culture combined with fura-2 microfluorimetry and pharmacological manipulation to determine the dependence of secretion on different types of Ca2+ channels. We stimulated cells with relatively long depolarizing voltage-clamp pulses in a medium containing 60 mm CaCl2. We found that, within a certain range of pulse parameters, secretion as measured by membrane capacitance changes was mainly determined by the total cumulative charge of Ca2+ inflow and the basal [Ca2+] level preceding a stimulus. Blocking or reducing the contribution of specific types of Ca2+ channels using either 20 μm nifedipine plus 10 μm nimodipine or 1 μmωCTxGVIA (omega-conotoxin GVIA) or 2 μmωCTxMVIIC (omega-conotoxin MVIIC) reduced secretion in proportion to Ca2+ charge, irrespective of the toxin used. We conclude that for long-duration stimuli, which release a large fraction of the readily releasable pool of vesicles, it is not so important through which type of channels Ca2+ enters the cell. Release is determined by the total amount of Ca2+ entering and by the filling state of the readily releasable pool, which depends on basal [Ca2+] before the stimulus. This result does not preclude that other stimulation patterns may lead to responses in which subtype specificity of Ca2+ channels matters.