1. Cloned large-conductance Ca(2+)-activated K+ channels from Drosophila (dslo) and human (hslo) were expressed in Xenopus oocytes. The effects of Ca2+ and voltage on these channels were compared by analysing both macroscopic currents and single-channel transitions. 2. The activation kinetics of dslo Ca(2+)-activated K+ channels are strongly influenced by the intracellular Ca2+ concentration, but are only minimally affected by membrane voltage. Current activation kinetics increase more than 60-fold in response to Ca2+ concentration increases in the range 0.56-405 microM, but increase less than 2-fold by voltage changes from -60 to +80 mV. 3. The activation kinetics of hslo channels are similarly influenced by increases in Ca2+ concentration; however, these kinetics are also increased 5- to 10-fold by voltage changes from -60 to +80 mV. 4. The deactivation kinetics of both dslo and hslo channels are also more Ca2+ sensitive than voltage sensitive. Increasing concentrations of Ca2+ slow deactivation kinetics more than 40-fold, while changes in the membrane voltage cause less than 2-fold changes. 5. Ca2+ increases the activation kinetics by altering first latency distributions. Increasing the Ca2+ concentration from 0.56 to 2.4 microM causes a 20-fold decrease in the mean time to first channel opening. 6. Both Ca2+ and voltage have large effects on regulating the steady-state open probability of these ion channels. Plots relating open probability (Po) to membrane voltage show a voltage dependence of 16.5 mV per e-fold change in Po for dslo and 12.3 mV per e-fold change in Po for hslo. At any given voltage the Ca2+ sensitivity of dslo is lower than that for hslo. The Hill coefficient for Ca2+ activation is 1.9 +/- 0.15, indicating that the binding of at least two Ca2+ ions is required to maximally activate both dslo and hslo channels. 7. The gating kinetics of both dslo and hslo channels can be well described by three open and five closed states. Changing the free Ca2+ concentration alters the time constants for the three longest closed states, without affecting any of the open states. Changing the membrane voltage alters the same three closed states, as well as the longest of the three open states. The two shortest occupancy open and closed time constants underlying these states are largely independent of voltage and Ca2+. 8. To account for these data, we propose that Ca2+ binding to the closed channel is the slow rate-limiting step in the activation pathway and, conversely, that Ca2+ unbinding is the slow rate-limiting step in the deactivation pathway. Hence, Ca2+ appears to bind to the closed channel and allows it to undergo a number of slow conformational changes that bring the channel to a state from which it can quickly open upon depolarization. These data imply that while both Ca2+ and voltage can alter the steady-state open probability of these channels, only Ca2+ has large effects on altering non-steady-state parameters and thus is the intracellular signal that predominantly modulates the rate of channel activation and deactivation.