Maitotoxin induces an extracellular Ca 2 + -dependent membrane depolarization predominantly via activation of L-type voltage-dependent Ca 2 + channels (L-VDCC) in GH 4 C 1 rat pituitary cells. In contrast to studies employing intracellular dyes, electrophysiological studies have indicated that maitotoxin activates voltage-independent conductances. In the present study, we used fura-2 calcium digital analysis to investigate the actions of very low concentrations of maitotoxin on cytosolic free calcium ([Ca 2 + ] i ) in GH 4 C 1 cells in an effort to distinguish different calcium entry mechanisms. Maitotoxin at concentrations as low as 0.01 ng/mL elevated [Ca 2 + ] i 35 ± 3% and induced membrane depolarization. The concentration dependency for maitotoxin-elevated [Ca 2 + ] i was biphasic with the first phase maximal at 0.05 to 0.5 ng/mL and the minimum ec 5 0 of the second phase about 2.0 ng/mL. Nimodipine (100 nM), a dihydropyridine antagonist of L-VDCC, prevented the [Ca 2 + ] i increase and depolarization induced by up to 0.1 ng/mL maitotoxin, but not at higher concentrations (0.5 ng/mL) of maitotoxin. This indicates that lower concentrations (0.1 ng/mL) of maitotoxin require L-VDCC, whereas higher concentrations ( 0.5 ng/mL) of maitotoxin may require additional ionic mechanisms. Maitotoxin (0.5 ng/mL) induced 4 5 Ca 2 + uptake and depolarization in L t k - cells which lack VDCC. Reducing extracellular Cl - from 123 to 5.8 mM increased the magnitude of membrane depolarization by maitotoxin (0.5 ng/mL), which suggests that a Cl - conductance participates in depolarization induced by higher maitotoxin concentrations. Taken together, our results indicate that maitotoxin activates at least two ionic mechanisms. At lower concentrations of maitotoxin, the primary ionic mechanism requires the activation of L-VDCC; however, at higher maitotoxin concentrations, additional ionic mechanisms are involved in the entry of extracellular Ca 2 + . This latter mechanism may represent the voltage-independent pathway evident under voltage clamp conditions.