Connexins form hemichannels at undocked plasma membranes and gap-junction channels (GJCs) at intercellular contacting zones. Under physiological conditions, hemichannels have low open probabilities, but their activation under pathological conditions, such as ischemia, induces and/or accelerates cell death. Connexin 46 (Cx46) is a major connexin of the lens, and mutations of this connexin induce cataracts. Here, we report the effects of linoleic acid (LA) on the electrical properties of Cx46 GJCs and hemichannels expressed in Xenopus laevis oocytes. LA has a biphasic effect, increasing hemichannel current at 0.1 μM and decreasing it at concentrations of 100 μM or higher. The effects of extracellular and microinjected LA conjugated to coenzyme A (LA-CoA) suggest that the current activation site is accessible from the intracellular but not extracellular compartment, whereas the current inhibitory site is either located in a region of the hemichannel pore inaccessible to intracellular LA-CoA, or requires crossing of LA through an organelle membrane. Experiments with other fatty acids demonstrated that the block of hemichannels depends on the presence of a hydrogenated double bond at position 9 and is directly proportional to the number of double bonds. Experiments in paired oocytes expressing Cx46 showed that LA does not affect GJCs. The block by unsaturated fatty acids reported here opens the possibility that increases in the concentration of these lipids in the lens induce cataract formation by blocking Cx46 hemichannels.