Ischemia-induced acidification of astrocytes or cardiac myocytes reduces intercellular communication by closing gap junction channels and subsequently internalizing gap junction proteins. To determine whether such coupling changes might be attributable to altered interactions between connexin43 (Cx43) and other proteins, we applied the nigericin/high K⁺ method to vary intracellular pH (pHi) in cultured cortical astrocytes. Intracellular acidification was accompanied by internalization of Cx43 with retention of Cx43 scaffolding protein Zonula Occludens-1 (ZO-1) at cell surfaces, suggesting that ZO-1 and Cx43 dissociate at low pHi. Coimmunoprecipitation studies revealed decreased binding of ZO-1 and increased binding of c-Src to Cx43 at low pHi. Resonant mirror spectroscopy was used to quantify binding of the SH3 domain of c-Src and the PDZ domains of ZO-1 to the carboxyl terminal domain of Cx43 (Cx43CT). Data indicate that the c-Src/Cx43CT interaction is highly pH dependent whereas the ZO-1/Cx43CT interaction is not. Moreover, binding of c-Src to Cx43CT prevented and reversed ZO-1/Cx43CT binding. We hypothesize that increased affinity of c-Src for Cx43 at low pHi aids in separation of Cx43 from ZO-1 and that this may facilitate internalization of Cx43. These data suggest that protracted acidification may remodel protein-protein interactions involving Cx43 and thus provide an important protective mechanism to limit lesion spread after ischemic injury.