Connexin46 (Cx46) participates in formation of the lens gap junctions which are a part of the lens transport system, devoted to maintenance of constant flow of nutrients in this avascular organ. Recent results showed that the D3Y mutation of human Connexin46 (hCx46) correlated with an autosomal dominant cataract. In our study we used two expression systems to analyse the physiological consequences of this mutation. Expression of EGFP labelled hCx46wt and hCx46D3Y in communication deficient HeLa cells showed that the mutation did not affect the capacity to form gap junction plaques. With dye transfer experiments we found that HeLa cells expressing the hCx46D3Y mutant revealed a higher degree of dye coupling compared to cells expressing the hCx46wt. Two-electrode voltage clamp experiments on Xenopus oocytes expressing hCx46wt and hCx46D3Y showed that hCx46wt formed voltage-sensitive hemichannels, which were not observed for the hCx46D3Y mutant. The reintroduction of a negatively charged residue, glutamic acid, at the third position generating the mutant hCx46D3E, restored the voltage-sensitivity of the resultant hemichannels in oocytes. Furthermore, HeLa cell pairs expressing hCx46D3E and the wild type showed a similar degree of dye coupling. We propose that the negatively charged aspartic acid at the third position of the N-terminus of hCx46 could determine the degree of metabolic cell-to-cell coupling and could play an important role for the voltage gating of hCx46 hemichannels.