Peroxisomal membrane proteins (PMPs) are essential components of the peroxisome that, depending on the specific protein, are essential for the organelle's biogenesis and/or maintenance. The 'Arabidopsis' 36 kilodalton peroxisomal membrane protein (PMP36) is a member of the ATP-binding-cassette family responsible for transporting small metabolic precursor molecules across the peroxisome boundary membrane. Previously, PMP36 was analysed in terms of its structure and expression during pumpkin seed germination, but has not been characterized in terms of its molecular targeting signals. Using tobacco plant suspension cells as an 'in vivo' import model system, PMP36 was determined to be situated in the peroxisomal membrane in an N matrix-Cmatrix topological orientation. In addition, two peroxisomal membrane targeting signals were identified within PMP36 and mutational analyses revealed that positively-charged amino acids within these mPTSs are required for efficient targeting to the peroxisome. Additional analyses revealed that PMP36 interacts with peroxin 19 isoform I (Pex19-I), a chaperone protein proposed to be responsible for the translocation of newly-synthesised PMPs to the peroxisomal membrane. Overall, these data indicate that PMP36 is a multi-spanning plant PMP with multiple targeting signals, and that PMP trafficking in plants maybe more complex than proposed in current working models of peroxisome biogenesis.