Peroxisomes perform a wide variety of metabolic processes in eukaryotic organisms. Mutations that affect peroxisome function or formation have profound phenotypic consequences, the latter demonstrated by peroxisome biogenesis disorders which are often fatal. The biogenesis of peroxisomes conceptually consists of: (1) the formation of the peroxisomal membrane, (2) the import of peroxisomal matrix enzymes and (3) the proliferation of the organelles. Proteins involved in these processes are collectively called peroxins, encoded by PEX-genes. To date 32 peroxins are known, which perform functions in peroxisome biogenesis that are conserved from yeast to man. In this article, we focus on the current status of knowledge about the topogenesis of the peroxisomal membrane proteins, and the import of proteins into the peroxisomal matrix.