Fatty acids are generally considered as agonists for peroxisome proliferator-activated receptors (PPARs). Fatty acids have been shown to bind to and transactivate PPARs; it is not known whether fatty acids act as generalized agonists for PPARs in different cell types, and thus, stimulate the expression of PPAR-regulated target genes. Here, we investigated the potency of unsaturated fatty acids on transactivation of PPRE, DNA-binding activity of PPARs, and the expression of a PPAR-regulated gene product, CD36. Docosahexaenoic acid (DHA) suppressed the basal and PPAR agonist-induced transactivation of PPRE, and DNA binding of PPARs in colon tumor cells (HCT116). The suppression of PPAR transactivation by DHA leads to reduced expression of CD36 in HCT116 cells and human monocytic cells (THP-1) as determined by promoter reporter gene assay and flow cytometric analysis. Our results demonstrate that DHA and other unsaturated fatty acids act as antagonists instead of agonists for transactivation of PPRE and PPAR-regulated gene expression in the cell lines tested. These results suggest that PPAR-mediated gene expression and cellular responses can be dynamically modulated by different types of dietary fatty acids consumed.