The peripheral myelin proteins P0 and PMP22 are associated in preparations of compact myelin and in cell cultures coexpressing both molecules. The mechanism of this interaction, however, still needs to be unravelled. We have established three different (cell-cell, cell-protein, protein-protein based) assay systems using retrovirally transduced HeLa cells that overexpressed either PMP22 or P0 and purified GST fusion oligopeptides of PMP22 and P0 to detect domain-specific interactions between these proteins. The results revealed that PMP22 and P0 are involved in both trans-homophilic and trans-heterophilic interactions. Moreover, the data clearly indicate that the heterophilic trans-interaction is mediated through the second loop of PMP22, while the first loop is involved in homophilic trans-interaction of PMP22 proteins. Both modes of interaction are due to direct protein-protein binding. In addition, we demonstrate that disease-related point mutations of P0 resulted in a decreased adhesion capability correlating with the severity of the respective disease phenotype.