Gap junctions are composed of connexins that form transmembrane channels between adjacent cells. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating. Interestingly, channel-independent processes regulated by Cx43 have also been postulated. In our studies to elucidate the mechanism of Cx43 channel gating by growth factors and to explore additional functions of gap junctions, we have identified three interacting partners of the C-terminal tail of Cx43 (Cx43CT). (i) the c-Src tyrosine kinase, which phosphorylates Cx43CT and is involved in G protein-mediated inhibition of Cx43 gap junctional communication. (ii) the ZO-1 'scaffold' protein, which might recruit signaling proteins into Cx43-based gap junctions. (iii) microtubules (consisting of alpha/beta-tubulin dimers), which extend with their distal ends to Cx43-based gap junctions, suggesting that Cx43 gap junctions may play a novel role in regulating microtubule stability in contacted cells. Here we show that Cx43 binds alpha-tubulin equally well as beta-tubulin. In addition, we show that the second, but not the first, PDZ domain of ZO-1 binds directly to Cx43, and we confirm that the very C-terminal isoleucine residue of Cx43 is critical for ZO-1 binding.