In the initiation of cardiac fibrosis the proliferation and differentiation of cardiac fibroblasts towards myofibroblasts are critical events, which might be inhibited by activation of PPARs. Therefore, we focused on the role of each PPAR isoform (PPARa, b/[delta] and [gamma]) in cardiac fibroblasts and myofibroblasts. Culturing and passaging neonatal rat cardiac fibroblasts on rigid culture discs induced a spontaneous transition from cardiac fibroblasts (passage 1-CF) to myofibroblasts (passage 3-CMF) as deduced from increased expression of a-smooth muscle actin and morphological changes. All three PPAR isoforms were found to be expressed in CF and CMF. However, PPARb/[delta] appeared to be functionally the most important isoform. Next, we observed that stimulation of PPARb/[delta], but not -a or -[gamma], with its ligand (GW501516) or adenoviral over expression significantly decreased the expression of a-smooth muscle actin in CF, indicating a reduced differentiation of CF to CMF. In addition, a BrdU incorporation assay indicated that the CF proliferation rate was inhibited ~40% only after treatment with PPARb/[delta] ligand. Moreover, the expression of G0S2, which is involved in growth arrest, was induced in both CF and CMF after stimulation of PPARb/[delta] with its ligand. Thus PPARb/d, but not -a or -[gamma], is involved in the inhibition of both cardiac fibroblast differentiation and proliferation.