The aim of the present study was to elucidate how transforming growth factor-beta(1) (TGF-beta(1)) can stimulate collagen deposition in cardiac tissue by interstitial cells via stimulation of fibroblasts, via myofibroblasts, or via differentiation of fibroblasts to myofibroblasts. The dose- and time-dependent stimulation of collagen production and of expression of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblasts, was studied in cultures of second-passage adult rat cardiac fibroblasts. The TGF-beta(1)-stimulated collagen production is positively correlated (r=0.68, P<0.001) with the appearance of alpha-SMA. Only at high concentrations (40 to 600 pmol/L) and after a long time (24 to 48 hours) of incubation, TGF-beta(1) increases the collagen production and stimulates the differentiation of fibroblasts to myofibroblasts. The maximal stimulation of the collagen production (2-fold, P<0.001) observed after incubation of cultures of fibroblasts with 600 pmol/L TGF-beta(1) for 48 hours is accompanied by a maximal stimulation of alpha-SMA expression (3.5-fold, P<0.001), when cultures consist mainly of myofibroblasts. The stimulation of collagen production cannot be reversed either after additional incubation of TGF-beta(1)-stimulated second-passage cultures for 2 days or in their offspring in the next third passage after incubation for 7 days without TGF-beta(1). The increased collagen production in these third-passage cultures cannot be further stimulated by TGF-beta(1). Our data suggest that TGF-beta(1)-stimulated collagen production in cultures of adult rat cardiac ventricular fibroblasts cannot be explained by a direct stimulation of the collagen production either in fibroblasts or in myofibroblasts. Instead, TGF-beta(1) induces the differentiation of fibroblasts to myofibroblasts, which have a higher activity for collagen production than fibroblasts.