Peroxisomes are important organelles of eukaryote cells. Although these structures are of relatively small size, they display an unprecedented functional versatility. The principles of their biogenesis and function are strongly conserved from very simple eukaryotes to humans. Peroxisome-borne proteins are synthesized in the cytosol and posttranslationally incorporated into the organelle. The protein-sorting signal for matrix proteins, peroxisomal targeting signal (PTS), and for membrane proteins (mPTS), are also conserved. Several genes involved in peroxisomal matrix protein import have been identified (PEX genes), but the details of the molecular mechanisms of this translocation process are still unclear. Here we describe procedures to study the subcellular location of peroxisomal matrix and membrane proteins in yeast and fungi. Emphasis is placed on protocols developed for the methylotrophic yeast Hansenula polymorpha, but very similar protocols can be applied for other yeast species and filamentous fungi. The described methods include cell fractionation procedures and subcellular localization studies using fluorescence microscopy and immunolabeling techniques.