Abstract: Two pathways have been identified for peroxisome formation: growth and division, and de novo synthesis. Recent experiments determined that peroxisomes originate at the endoplasmic reticulum (ER). Although many proteins have been implicated in the peroxisome biogenic program, no proteins in the eukaryotic secretory pathway have been identified as having roles in peroxisome formation. Using the yeast Saccharomyces cerevisiae regulatable Tet-promoter Hughes clone collection, we found repression of the ER-associated secretory proteins, Sec20p and Sec39p, resulted in mislocalization of the peroxisomal matrix protein chimera Pot1p-GFP to the cytosol. Likewise, the peroxisomal membrane protein chimera Pex3p-GFP localized to tubular-vesicular structures in cells suppressed for Sec20p, Sec39p and Dsl1p, which form a complex at the ER. Loss of Sec39p attenuated formation of Pex3p-derived peroxisomal structures following galactose induction of Pex3p-GFP expression from the GAL1 promoter. Expression of Sec20p, Sec39p and Dsl1p were moderately increased in yeast grown under conditions that proliferate peroxisomes, and Sec20p, Sec39p and Dsl1p were found to cofractionate with peroxisomes and colocalize with Pex3p-mRFP under these conditions. Our results show that SEC20, SEC39 and DSL1 are essential secretory genes involved in the early stages of peroxisome assembly and are the first to identify and characterize an ER-associated secretory machinery involved in peroxisome biogenesis.