OBJECTIVE In order to isolate voltage-gated calcium currents in rods retaining intact axons and presynaptic terminals, it is first necessary to identify specific blockers of the large calcium-dependent chloride current, ICl(Ca), which obscures them. Based upon previous reports of its efficacy as an inhibitor of a volume regulated chloride channel (VRAC), a calcium-dependent chloride channel, and the cystic fibrosis transmembrane conductance regulator (CFTR), we investigated whether the serotonin reuptake inhibitor, fluoxetine hydrochloride, could act as a specific blocker for ICl(Ca) in salamander rod photoreceptor terminals, without affecting other aspects of rod physiology. METHODS Intact rod photoreceptors retaining axons and presynaptic terminals were enzymatically dissociated from salamander retinae. Under whole cell voltage clamp, depolarization-induced whole cell currents were recorded in the presence and absence of fluoxetine (10 and 50 microM pipette concentration) administered via a puffing pipette or in the bath (25 microM). Changes in intracellular free calcium levels were monitored as changes in fura-2 fluorescence following brief depolarization with high K+ (50 and 100 mM) administered via a puffing pipette in the presence and absence of fluoxetine (4 and 10 microM) in the bath. RESULTS When puffed onto cells, fluoxetine inhibited ICl(Ca) in a dose-dependent fashion (50 microM=96% reduction; 10 microM=14% reduction). In addition to the reduction in amplitude of ICl(Ca), 4 microM fluoxetine (pipette concentration) significantly reduced the duration of ICl(Ca) (48% reduction). Fluoxetine also suppressed the calcium-activated potassium current, IK(Ca), to similar extents (50 microM=75% reduction; 10 microM=23% reduction) when puffed onto cells. Preincubation of rods with 25 microM fluoxetine in the bath significantly reduced outward currents at both 0 mV, where ICl(Ca) is negligible because ECl is about 0 mV and the bulk of the current is carried by IK(Ca), and at +40 mV, where the current is a combination of ICl(Ca) and IK(Ca). Parallel calcium imaging experiments with fura-2 revealed that preincubation of rods in 10 microM fluoxetine virtually eliminated the normal rise in intracellular free calcium in somatic (99.6% reduction) and terminal (98% reduction) compartments following brief depolarization with high K+ (100 mM pipette concentration). Cells preincubated in 4 microM fluoxetine, a therapeutically relevant concentration, showed smaller but significant reductions in Ca2+ elevations in both somatic (66% reduction) and terminal (36% reduction) compartments and even more significant reductions in the duration of sustained calcium levels of the terminal compartment (50% reduction) following brief depolarization with high K+ (50 mM pipette concentration). CONCLUSIONS We conclude that in addition to blocking ICl(Ca), fluoxetine inhibits IK(Ca). We further conclude that the inhibition of both of these currents is the consequence of inhibition of the normal sustained elevation in intracellular calcium following depolarization and initial calcium influx. Combined, the data suggest that fluoxetine may have multiple sites of action in rod photoreceptors instead of acting as a specific inhibitor of ICl(Ca).