Communication across gap junctions between cells in various tissues is considered an important mechanism for control of cellular growth, differentiation and function. Although cell-cell coupling in the gill epithelium is likely for functional reasons, the common view is that gap junctions are not present between cells in the gill epithelium of teleostean fish. Gap junction mediated cell-cell communication was analysed in the present study in primary and secondary cultures of rainbow trout gill epithelial cells by microinjecting the fluorescent dye Lucifer yellow CH. In 4–14 day-old primary and secondary cell cultures, 58% of the cells injected with Lucifer yellow were coupled to at least one other cell. To exclude the possibility that intercellular dye transfer occurred through cytoplasmic bridges instead of gap junctions, we also microinjected FITC-dextran, which because of its molecular size does not pass through gap junctions. None of the cells injected with FITC-dextran showed spreading of the dye. The number of cells spreading Lucifer yellow was decreased in a dose-dependent way when the cells were treated with dioctanoylglycerol, a synthetic diacylglycerol known to affect the open state of gap junctions, which further supports that the cells are coupled through functional gap junctions.