Older clots become less sensitive to fibrin degradation than newly formed ones. A possible role for fibroblasts in this defective thrombus lysis was studied. A system has been developed in which different clones of fibroblasts were incorporated into a floating whole blood clot. The effect of the incorporated fibroblasts on clot lysis has been analyzed in relation to their basic characteristics: clot retraction, production of plasminogen activators (PAs) and their inhibitors (PAIs), and secretion of collagen. In neoplastic fibroblast-enriched clots, secretion of PA was associated with spontaneous lysis of a whole blood clot. Normal fibroblasts, secreting levels of PA and PAI similar to those of the cancer cells, did not induce spontaneous lysis of the clot. Moreover, these cells protected whole blood clot from thrombolysis by added PA. Our data show that the resistance to fibrin clot degradation induced by normal fibroblasts was mainly mediated by collagen secretion and deposition rather than PAI secretion or retraction of the clot. We suggest a key role for normal fibroblasts in the acquisition of resistance to proteolytic fibrin degradation of whole blood clots through the secretion of collagen.