Apoptosis of fibroblasts may be key for the removal of cells following repair processes. Contraction of three-dimensional collagen gels is a model of wound healing and remodeling. Here two potent inducers of contraction, TGF-beta1 and fetal calf serum (FCS) were evaluated for their effect on fibroblast apoptosis in contracting collagen gels. Human fetal lung fibroblasts were cultured in floating type I collagen gels, exposed to TGF-beta1 or FCS, and allowed to contract for 5 days. Apoptosis was evaluated using TUNEL and confirmed by DNA content profiling. Both TGF-beta1 and serum significantly augmented collagen gel contraction. TGF-beta1 also increased apoptosis assessed by TUNEL positivity and DNA content analysis. In contrast, serum did not affect apoptosis. TGF-beta1 induction of apoptosis was associated with augmented expression of Bax, a pro-apoptotic member of the Bax/Bcl-2 family, inhibition of Bcl-2, an anti-apoptotic member of the same family, and inhibition of both cIAP-1 and XIAP, two inhibitors of the caspase cascade. Serum was associated with an increase in cIAP-1 and Bcl-2, anti-apoptotic proteins. Interestingly, serum was also associated with an apparent increase in Bax, a pro-apoptotic protein. Blockade of Smad3 with either siRNA or by using murine fibroblasts deficient in Smad3 resulted in a lack of TGF-beta induction of augmented contraction and apoptosis. Contraction induced by different factors, therefore, may be differentially associated with apoptosis, which may be related to the persistence or resolution of the fibroblasts that accumulate following injury.