Structure/function analysis shows that the carboxyl terminal (CT) domain of connexin43 (Cx43) is essential for the chemical regulation of cell-cell communication. Of particular interest is the region between amino acids 260 and 300. Structural preservation of this region is essential for acidification-induced uncoupling (ie, pH gating). In this study, we report data showing that a 17mer peptide of the same sequence as amino acids 271 to 287 of Cx43 (CSSPTAPLSPMSPPGYK) can prevent pH gating of Cx43-expressing oocytes. Experiments were carried out in pairs of Xenopus oocytes previously injected with connexin38 antisense and expressing wild-type Cx43. Junctional conductance was measured electrophysiologically. pHi was determined from the light emission of the proton-sensitive dye dextran-seminaphthorhodafluor. Intracellular acidification was induced by superfusion with a bicarbonate-buffered solution gassed with a progressively increasing concentration of CO2. Injection of water alone into both oocytes of a Cx43-expressing pair or injection of a peptide from region 321 to 337 of Cx43 did not modify pH sensitivity. However, injection of a polypeptide corresponding to amino acids 241 to 382 of Cx43 interfered with the ability of gap junctions to close on acidification. Similar results were obtained when a 17mer peptide (region 271 to 287) was injected into both oocytes of the pair. Normal Cx43 pH gating was observed if (1) the amino acid sequence of the 17mer peptide was scrambled or (2) the N and the C ends of the 17mer peptide were not included in the sequence. This is the first demonstration of a molecule that can interfere with the chemical regulation of connexin channels in a cell pair. The data may lead to the development of small molecules that can be used in Cx43-expressing multicellular preparations to study the role of gap junction regulation in normal as well as diseased states.