Gap junctions are intercellular channels that form a direct link between the cytoplasm of two adjacent cells. Gap junctional intercellular communication (GJIC) is long known to be necessary for the passive diffusion of small molecules like ions, cAMP, IP3 and calmodulin and has recently been found to be involved in complex processes like progenitor cell differentiation and maintenance of stemness in embryonic stem cells. In the current study we investigated whether cells can exchange miRNAs through gap junctions. miRNAs are small dsRNA molecules that are able to exert post-translational fine-regulation in cellular gene expression. HeLa cells served as a model organism in our experiments. In the wild-type (HeLa-wt) they are deficient in the connexins needed to form functional gap junctions. Transfectants stably expressing connexin-43 (HeLa-43) but not HeLa-wt exhibited GJIC through the transfer of the small, membrane impermeable fluorescent dye calcein between co-cultured cells (n=6, p<0.05). To investigate the transfer of miRNA we also used a co-culture approach. HeLa-wt and HeLa-43 served as donor cells and were transfected with synthetic miRNA (50 nM) for miR-124. miR-124 is a well characterised neuronal miRNA that is known to be only weakly expressed in HeLa. HeLa-43 detector cells were fluorescently labelled with a stable membrane dye and transfected with a dual-luciferase reporter system. For this purpose renilla luciferase was put under control of a miR-124 sensitive 3' UTR and firefly luciferase served as an internal expression control. Donor and detector cells were then co-cultured for five days followed by FACS separation of the two populations. Luciferase activity was quantified using a luminometer and renilla activity normalised to firefly luciferase. In detector cells co-cultured with HeLa-43 donor cells renilla luciferase activity was significantly downregulated by 30 +/­15% as compared to cells co-incubated with HeLa-wt donor cells (n=5, p<0.05). In control cells being directly transfected with miR-124 and the luciferase system, the reduction in renilla luciferase activity was around 80% (n=3, p<0.05). Our results show efficient transfer of miR-124 between HeLa cells coupled via Cx43-containing gap junctions resulting in miR-124 dependent gene silencing. Transfer of miRNA between cells through gap junctions is a potential new way to control gene programmes in adjacent cells during development and functional maturation of cellular networks.