Lens fiber connexins, cx50 and cx46 (alpha3 and alpha8), belong to a small subset of connexins that can form functional hemichannels in nonjunctional membranes. Knockout of either cx50 or cx46 results in a cataract, so the properties of both connexins are likely essential for proper physiological functioning of the lens. Although portions of the sequences of these two connexins are nearly identical, their hemichannel properties are quite different. Cx50 hemichannels are much more sensitive to extracellular acidification than cx46 hemichannels and differ from cx46 hemichannels both in steady-state and kinetic properties. Comparison of the two branches of the cx50 hemichannel G-V curve with the junctional G-V curve suggests that cx50 gap junctions gate with positive relative polarity. The histidine-modifying reagent, diethyl pyrocarbonate, reversibly blocks cx50 hemichannel currents but not cx46 hemichannel currents. Because cx46 and cx50 have very similar amino acid sequences, one might expect that replacing the two histidines unique to the third transmembrane region of cx50 with the corresponding cx46 residues would produce mutants more closely resembling cx46. In fact this does not happen. Instead the mutant cx50H161N does not form detectable hemichannels but forms gap junctions indistinguishable from wild type. Cx50H176Q is oocyte lethal, and the double mutant, cx50H61N/H176Q, neither forms hemichannels nor kills oocytes.