Although regulation of chemical transmission is known to involve the interaction of receptors with scaffold proteins, little is known about the existence of protein-protein interactions in regulating gap junction-mediated electrical synapses. The scaffold protein zonula-occludens-1 (ZO-1), a member of the MAGUK family of proteins, was reported to interact with several connexins (Cxs). We show here that ZO-1 extensively colocalizes with Cx35 at identifiable "mixed" (electrical and chemical) contacts on goldfish Mauthner cells, a model synapse for the study of vertebrate electrical transmission where it is possible to correlate physiological properties with molecular composition. Further, our analysis indicates that these proteins directly interact at goldfish electrical synapses. In contrast to Cx43, which interacts with ZO-1 via the PDZ2 domain, Cx35 interacts with ZO-1 via the PDZ1 domain, and this association is of lower affinity. The properties of the ZO-1/Cx35 association suggest the existence of a more dynamic relation between these two proteins, possibly including a role of ZO-1 in regulating gap junctional conductance at these highly modifiable electrical synapses. The interaction of ZO-1 with conserved regions of the C termini of Cx35/Cx36 orthologs may have a common function at electrical synapses of mammals and other vertebrates.