The objective of this study was to investigate the presence of Thy-1 in the myocardium and on cardiac fibroblasts and to determine whether or not cardiac fibroblasts form a heterogeneous population in term of Thy-1 expression. Thy-1 expression was examined by immunohistology of ventricular sections from normal and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Thy-1 immunostaining was detected in connective tissue on α8 integrin-positive and discoidin domain receptor 2 (DDR2)-positive fibroblasts. Enhanced Thy-1 staining was observed in the hearts of DOCA-salt rats particularly in areas of interstitial fibrosis. Cardiac mRNA analysis confirmed the increased Thy-1 expression. On cultured cardiac fibroblasts, flow cytofluorometry showed that cells, from primary culture to passage 4, were double positive for Thy-1 and for both α8 integrin and DDR2. Analysis of isolated lipid rafts by detergent-free sucrose gradient indicated that Thy-1 protein was probably located in these structures, but it may be located on a membrane microdomain slightly different from those of caveolin-1, as revealed by immunocytochemistry. Differentiation of fibroblasts into myofibroblasts being a characteristic of cardiac fibrosis and scarring, cardiac fibroblasts were stimulated in the presence of transforming growth factor-beta (TGF-β) or connective tissue growth factor. While the expression of smooth muscle α-actin and α8 integrin doubled, Thy-1 level, measured by Western blotting and flow cytofluorometry, was not influenced by TGF-β. These results demonstrate that cardiac fibroblasts express Thy-1 and form a homogeneous population. Thy-1 expression also appears to be independent of fibroblast differentiation. The dichotomy between the increased Tthy-1 expression in the fibrotic area and the lack of association with fibroblast differentiation suggests that Thy-1 may represent a marker of fibroblast proliferation in the myocardium.