Peroxisomes are subcellular organelles found virtually in all eukaryotic cells. They perform various functions depending on organism, cell type or environmental conditions. The importance of peroxisomes for human development and health is shown by the occurrence of inherited peroxisomal disorders. Peroxisome biogenesis involves a number of processes including peroxisomal membrane protein and matrix protein import, growth of the lipid bilayer and proliferation. Peroxisomal matrix proteins are directed to peroxisomes by the conserved targeting signals PTS1 and PTS2. However, a subset of matrix proteins neither contains a PTS1 nor PTS2. In this, study I describe how one of these latter enzymes, the nicotinamidase Pnc1 enters peroxisomes. We found that Pnc1 is co-imported with the PTS2-containing enzyme Gpd1 by a piggy-back mechanism. This mechanism requires the PTS2 receptor Pex7 and its coreceptor Pex21. In addition, we found that Pnc1 piggy-back import relies on homodimerisation of Gpd1. In the second part of this thesis, I describe the analysis of the AAA+ATPase Msp1 and the attempts to uncover its role in peroxisome biogenesis. A genome wide screen revealed a genetic interaction with two proteins; Pex25 and the novel PMP Ygr168c indicating a role of Msp1 in the regulation of peroxisome abundance. A preliminary analysis of Ygr168c is also presented. Together these studies further our understanding of peroxisomal protein import and biogenesis and provide novel areas for future exploration.