The molecular mechanisms controlling pH-sensitivity of gap junctions formed of two different connexins are yet to be determined. We used a proton-sensitive fluorophore and electrophysiological techniques to correlate changes in intracellular pH (pH i ) with electrical coupling between connexin-expressing Xenopus oocytes. The pH sensitivities of ␣ 3 (connexin46), ␣ 2 (connexin38), and ␣ 1 (connexin43) were studied when these proteins were expressed as: 1) nonjunctional hemichannels (for ␣ 3 and ␣ 2 ), 2) homotypic gap junctions, and 3) heterotypic gap junctions. We found that ␣ 3 hemichannels are sensitive to changes in pH i within a physiological range (pKa ϭ 7.13 Ϯ 0.03; Hill coefficient ϭ 3.25 Ϯ 1.73; n ϭ 8; mean Ϯ SEM); an even more alkaline pKa was obtained for ␣ 2 hemichannels (pKa ϭ 7.50 Ϯ 0.03; Hill coefficient ϭ 3.22 Ϯ 0.66; n ϭ 13). The pH sensitivity curves of ␣ 2 and ␣ 3 homotypic junctions were indistinguishable from those recorded from hemichannels of the same connexin. Based on a comparison of pKa values, both ␣ 3 and ␣ 2 gap junctions were more pH i -dependent than ␣ 1 . The pH sensitivity of ␣ 2 -containing heterotypic junctions could not be predicted from the behavior of the two connexons in the pair. When ␣ 2 was paired with ␣ 3 , the pH sensitivity curve was similar to that obtained from ␣ 2 homotypic pairs. Yet, pairing ␣ 2 with ␣ 1 shifted the curve similar to homotypic ␣ 1 channels. Pairing ␣ 2 with a less pH sensitive mutant of ␣ 1 (M257) yielded the same curve as when ␣ 1 was used. However, the pH sensitivity curve of ␣ 3 /␣ 1 channels was similar to ␣ 3 /␣ 3 , while ␣ 3 /M257 was indistinguishable from ␣ 3 /␣ 1 . Our results could not be consistently predicted by a probabilistic model of two independent gates in series. The data show that dissimilarities in the pH regulation of gap junctions are due to differences in the primary sequence of connexins. Moreover, we found that pH regulation is an intrinsic property of the hemichannels, but pH sensitivity is modified by the interactions between connexons. These interactions should provide a higher level of functional diversity to gap junctions that are formed by more than one connexin. Dev.