The protein import machinery of the peroxisome consists of many proteins, collectively called the peroxins. By applying the split-ubiquitin technique we systematically tested the pair-wise interactions between the Nub- and Cub-labeled peroxins for the first time in the living cells of the yeast Saccharomyces cerevisiae. We found that Pex10p plays a central role in the protein interaction network by connecting the ubiquitin conjugation enzyme Pex4p to the other members of the protein import machinery. A yeast strain harboring a deletion of PEX3 enabled us to estimate the influence of the peroxisomal membrane on the formation of a subset of the investigated protein-protein interactions.