Protein tyrosine dephosphorylation by PTP1B is an important step in the regulation of many cell signaling cascades. While PTP1B itself is anchored with its C-terminus to the ER membrane network, many of its substrates are localized to the plasma membrane. This spatial separation raises the question how PTP1B can find its targets. In our study we used the method of Bimolecular Fluorescence Complementation (BiFC) (Hu et al., 2002, Mol. Cell, 9:789–98) to demonstrate direct protein-protein interactions between PTP1B and PKCdin vivo. PKC[delta] is a cytosolic protein which in the presence of PMA translocates to the plasma membrane. With the BiFC technique we could demonstrate that in resting cells PKC[delta] forms complexes with PTP1B at the ER membrane network. When the cells were treated with PMA the BiFC signal increased and a strong accumulation of PTP1B/PKC[delta] complexes within large spots at the cell border could be observed. This PMA-induced spot formation could not be seen when the C-terminal ER membrane anchor of PTP1B was removed. Complexes of PKC[delta] with such C-terminally truncated PTP1Bs were, in the presence of PMA, homogenously accumulated all over the plasma membrane. Therefore, our data indicate that a direct interaction between ER-anchored and plasma membrane-anchored proteins is possible.