Previously it has been shown that the endogenous Arabidopsis peroxin, AtPEX16, coexisted at steady state in membranes of the endoplasmic reticulum (ER) and peroxisomes. Herein, an ER-to-peroxisome trafficking pathway and the requisite molecular targeting signals for mycAtPEX16 transiently expressed in Arabidopsis and tobacco BY-2 suspension cells are described. Immunofluorescent mycAtPEX16 was observed initially in the cytosol (<2 h) and subsequently (2-4 h) in perinuclear/reticular ER and non-Golgi/non-peroxisome structures termed the ER-peroxisome intermediate compartment. After 4 h, all catalase- and ascorbate peroxidase-containing peroxisomes also possessed mycAtPEX16, indicative of mycAtPEX16 sorting to pre-existing peroxisomes. Incubations of bombarded cells at 15 degrees C, or in brefeldin A at 25 degrees C, resulted in accumulations of mycAtPEX16 within the ER. Following re-equilibration of cold-treated cells at 25 degrees C, or removal of brefeldin A, mycAtPEX16 was observed mainly in the ER-peroxisome intermediate compartment, and later within all of the peroxisomes in both species. Two internal membrane helices and the intervening sequence including the amino acid residues -VRS- were found necessary and sufficient for targeting AtPEX16 first to the ER and then to peroxisomes. Individual targeting signals for these organelles were indistinguishable, indicative of overlapping signal(s). In summary, the trafficking study of AtPEX16 revealed a dynamic link between the ER and pre-existing peroxisomes, which provided novel data in support of an upgraded semi-autonomous peroxisome model portraying participation of the ER in the sorting of certain peroxisome membrane proteins, such as AtPEX16, through an intermediate compartment to pre-existing plant peroxisomes.