Peroxisome ghosts are aberrant peroxisomal structures found in cultured skin fibroblasts from patients affected by Zellweger Syndrome (ZS), a genetic disorder of peroxisomal assembly. They contain peroxisomal integral membrane proteins (PxIMPs) and they lack most of the matrix enzymes that should be inside the organelle (Santos et al., Science 239 (1988) 1536-1538). Considerable evidence indicates that these ghosts result from genetic defects in the cellular machinery for importing newly-synthesized peroxisomal proteins into the organelle. In contrast to these observations, (Heikoop et al., Eur. J. Cell Biol. 57 (1992) 165-171) report that in Zellweger Syndrome, peroxisomal membranes are located within lysosomes and/or contain lysosomal enzymes. We have undertaken a more detailed and systematic investigation of this matter, employing confocal laser scanning microscopy (CLSM). In fibroblasts derived from ZS patients belonging to different complementation groups, peroxisomes were labeled with antibodies against PxIMPs and lysosomes were labeled with an antibody against a lysosome associated membrane protein (LAMP-2) or with LysoTracker. The results unambiguously demonstrated no appreciable colocalization of PxIMPs and LAMPs (or LysoTracker), indicating that peroxisomal ghosts are distinct subcellular structures, occupying separate subcellular locations.