The peroxisome proliferator-activated receptor α (PPARα) plays a key role in lipid and lipoprotein metabolism. However, important inter- and intraspecies differences exist in the response to PPARα activators. This incited us to screen for PPARα variants with different signaling functions. In the present study, using a RT-PCR approach a variant human PPARα mRNA species was identified, which lacks the entire exon 6 due to alternative splicing. This deletion leads to the introduction of a premature stop codon, resulting in the formation of a truncated PPARα protein (PPARα_tr) lacking part of the hinge region and the entire ligand-binding domain. RNase protection analysis demonstrated that PPARα_tr mRNA is expressed in several human tissues and cells, representing between 20–50% of total PPARα mRNA. By contrast, PPARα_tr mRNA could not be detected in rodent tissues. Western blot analysis using PPARα-specific antibodies demonstrated the presence of an immunoreactive protein migrating at the size of in vitro produced PPARα_tr protein both in human hepatoma HepG2 cells and in human hepatocytes. Both in the presence or absence of 9-cis-retinoic acid receptor, PPARα_tr did not bind to DNA in gel shift assays. Immunocytochemical analysis of transfected CV-1 cells indicated that, whereas transfected PPARα_wt was mainly nuclear localized, the majority of PPARα_tr resided in the cytoplasm, with presence in the nucleus depending on cell culture conditions. Whereas a chimeric PPARα_tr protein containing a nuclear localization signal cloned at its N-terminal localized into the nucleus and exhibited strong negative activity on PPARα_wt transactivation function, PPARα_tr interfered with PPARα_wt transactivation function only under culture conditions inducing its nuclear localization. Cotransfection of the coactivator CREB-binding protein relieved the transcriptional repression of PPARα_wt by PPARα_tr, suggesting that the dominant negative effect of PPARα_tr might occur through competition for essential coactivators. In addition, PPARα_tr interfered with transcriptional activity of other nuclear receptors such as PPARγ, hepatic nuclear factor-4, and glucocorticoid receptor-α, which share CREB-binding protein/p300 as a coactivator. Thus, we have identified a human PPARα splice variant that may negatively interfere with PPARα_wt function. Factors regulating either the ratio of PPARα_wtvs. PPARα_tr mRNA or the nuclear entry of PPARα_tr protein should therefore lead to altered signaling via the PPARα and, possibly also, other nuclear receptor pathways.