Phthalate esters belong to a large class of compounds known as peroxisome proliferators (PP). PP include chemicals that activate different subtypes of the peroxisome proliferator-activated receptor (PPAR) family. The ability of phthalate esters and their metabolites to activate responses through different PPAR subtypes is not fully characterized. We investigated the ability of two phthalate esters di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) and selected metabolites to activate PPAR (alpha, beta/delta, gamma) using a transient transfection assay. The monoester of DEHP, mono-(2-ethylhexyl) phthalate (MEHP) activated all three subtypes of PPAR, but preferentially activated PPARalpha. A second metabolite of DEHP, 2-ethylhexanoic acid (2-EHXA) was a weaker activator of all three subtypes. DBP, but not the primary metabolite mono-n-butyl phthalate weakly activated all three PPAR subtypes. MEHP and DBP but not DEHP and MBP interacted directly with human PPARalpha and PPARgamma as determined by scintillation proximity assays. Both DEHP and DBP activated expression of PP-inducible gene products in wild-type but not PPARalpha-null mice suggesting that both of these phthalates exert their effects by activation of PPARalpha in vivo. The preferential activation of PPARalpha by phthalate ester metabolites suggests that these phthalates mediate their toxic effects in rodent liver in a manner indistinguishable from other PP.