Hemi-gap-junction (HGJ) channels of retinal horizontal cells (HCs) function as transmembrane ion channels that are modulated by voltage and calcium. As an endogenous retinal neuromodulator, zinc, which is co-released with glutamate at photoreceptor synapses, plays an important role in shaping visual signals by acting on postsynaptic HCs in vivo. To understand more fully the regulation and function of HC HGJ channels, we examined the effect of Zn²⁺ on HGJ channel currents in bass retinal HCs. Hemichannel currents elicited by depolarization in Ca²⁺-free medium and in 1 mM Ca²⁺ medium, were significantly inhibited by extracellular Zn²⁺. The inhibition by Zn²⁺ of hemichannel currents was dose-dependent with a half-maximum inhibitory concentration of 37 µM. Compared with other divalent cations, Zn²⁺ exhibited higher inhibitory potency with the order being Zn²⁺ > Cd²⁺ Co²⁺ > Ca²⁺ > Ba²⁺ > Mg²⁺. Zn²⁺ and Ca²⁺ were found to modulate HGJ channels independently in additivity experiments. Modification of histidine residues with N-bromosuccinimide (N-BrSuc) suppressed the inhibitory action of Zn²⁺, while modification of cysteine residues had no significant effect on Zn²⁺ inhibition. Taken together, these results suggest that zinc acts on HGJ channels in a calcium-independent way, that histidine residues on the extracellular domain of HGJ channels mediate the inhibitory action of zinc.