The peroxisome is an organelle found in most eukaryotes that is crucial for lipid metabolism. The ability of peroxisomes to divide themselves and transport post-translational proteins suggests that the peroxisome may have had an endosymbiotic origin. However, the localization of peroxisomal proteins to the endoplasmic reticulum (ER) and the similarity of some peroxisomal proteins to those localized in the ER suggest an alternative hypothesis: that the peroxisome was developed from the ER. To study the evolutionary distance between the peroxisome, the ER and prokaryotes, we conducted a phylogenetic analysis of cell division control 48 (CDC48) and its homologs, including the ER-localized CDC48, the CDC48 homologs in prokaryotes and the peroxisome-localized PEX1 and PEX6. We also conducted a similarity search of peroxisomal protein sequences against prokaryotic protein sequences using BLAST at several E-value thresholds. We provide several lines of evidence supporting an actinobacteria symbiotic origin for the peroxisome: (1) PEX1 and PEX6 are more closely related to the CDC48 homologs in actinobacteria than to the ER-localized CDC48; (2) actinobacterial proteins show higher levels of similarity to those of the peroxisome than to those of other prokaryotes.