The role of the Saccharomyces cerevisiae Pip2p-Oaf1p transcription factor was examined in reference to the regulation of the peroxin gene PEX25 involved in peroxisome proliferation. The PEX25 promoter contains an oleate response element (ORE)-like sequence comprising a CGG palindrome lacking a canonical adenine, which is considered critical for element function and Pip2p-Oaf1p binding. Pex25p levels were higher in wild-type cells grown on oleic acid medium than in those grown on ethanol, but this induction was abolished in cells devoid of Pip2p-Oaf1p. Studies based on lacZ reporter genes and in vitro protein-DNA interactions revealed that the PEX25 ORE could bind Pip2p-Oaf1p and confer activation on a basal promoter. These findings reinforced the central role played by Pip2p-Oaf1p in regulating peroxisome proliferation. We also investigated whether Pip2p-Oaf1p is important for regulating genes encoding peroxins involved in protein import into the peroxisomal matrix. Pip2p-Oaf1p was able to bind efficiently to the PEX5 ORE but not to an ORE-like CGG palindrome in the PEX14 promoter. However, immunoblotting revealed that both Pex5p and Pex14p (as well as Pex7p and Pex13p) were not more abundant in cells grown on oleic acid medium compared with ethanol. These data on a functional, adenine-less, PEX25 ORE and a nonfunctional N13-spaced ORE-like sequence in the PEX14 promoter capable of binding Pip2p-Oaf1p prompts readjustment of the ORE consensus to comprise CGGN3TNA/(R)N8-12CCG.