14-3-3 proteins are intracellular adapter molecules which are involved in the regulation of cell signalling, in cell cycle control and in apoptosis. In human cells there are seven isoforms (b, [gamma], e, [zeta], [eta], [sigma] and [tau]) which form both, homo- and heterodimers. With their phosphoserine/phosphothreonine binding sites, 14-3-3 proteins can interact with many other proteins. In our study we isolated all human 14-3-3 isoforms from HEK293-cells and produced YFP fusion proteins to investigate the intracellular localization of the 14-3-3 proteins. The homologous interaction among 14-3-3 proteins as well as heterologous binding to other proteins was studied with the technique of bimolecular fluorescence complementation (BiFC) (Hu et al., 2002, Mol. Cell, 9:789–98). This method is based on the molecular complementation of two non-fluorescent fragments of YFP (YFP1 and YFP2) fused to interacting partners. We generated fusion-proteins of each 14-3-3 isoform, which contained either YFP1 or YFP2 at the C- or N-terminus. With the BiFC method we could demonstrate homo- and heterologous dimerization of 14-3-3 isoforms in vivo. Furthermore, we could show heterologous binding of 14-3-3 proteins to the protein kinases PKCd and src, to the protein phosphatase PTP1B and to actin.