The 22-kDa peroxisomal membrane protein (PMP22) is a major component of peroxisomal membranes in mammals. Although its precise role in peroxisome function is poorly understood, it seems to be involved in pore forming activity and may contribute to the unspecific permeability of the organelle membrane. PMP22 is synthesized on free cytosolic ribosomes and then directed to the peroxisome membrane by specific targeting information. Previous studies in rats revealed that PMP22 contains one distinct peroxisomal membrane targeting signal in the amino-terminal cytoplasmic tail. We cloned and characterized the targeting signal of human PMP22 and compared it with the already described characteristics of the corresponding rat protein. Amino acid sequence alignment of rat and human protein revealed 77% identity including a high conservation of several protein motifs. We expressed various deletion constructs of PMP22 in fusion with the green fluorescent protein in COS-7 cells and determined their intracellular localization. In contrast to previous studies on rat PMP22 and most other peroxisomal membrane proteins, we showed that human as well as rat PMP22 contains two distinct and nonoverlapping peroxisomal membrane targeting signals, one in the amino-terminal and the other in the carboxyl-terminal protein region. They consist of two transmembrane domains and adjacent protein loops with almost identical basic clusters. Both of these peroxisomal targeting regions interact with PEX19, a factor required for peroxisome membrane synthesis. In addition, we observed that fusing the green fluorescent protein immediately adjacent to the targeting region completely abolishes targeting function and mislocalizes PMP22 to the cytosol.