Objective: To study fibroblasts and mast cells in human peritoneal adhesions and to evaluate whether their interaction plays a role in adhesion development. Summary Background Data: Myofibroblasts play a critical role in wound repair/fibrosis. Mast cells influence the formation of peritoneal adhesions in a rat model, and they are modulators of fibroblast functions. Methods: Peritoneal adhesion biopsies were processed for either histology (H&E, toluidine blue) or immunohistochemistry (tryptase, laminin, collagen type IV and VIII, and α-SMA) or grown as explants for obtention of fibroblasts. The effects of mast cell (HMC-1) sonicate and selected mast cell mediators and cytokines on fibroblast proliferation ([ 3H]thymidine) and collagen synthesis ([ 3H]proline) and on fibroblast contractile activity (tri-dimensional collagen lattice) were evaluated. Mast cell mediators: influencing fibroblast proliferation were partially characterized by enzymatic susceptibility and FPLC gel filtration column chromatography. Results: Most of the fibroblasts in peritoneal adhesions were identified as α-SMA-positive myofibroblasts. Mast cell hyperplasia was observed and more than one third of the mast cells were degranulated. Few mast cells showed a faint staining for laminin or collagen type IV and VIII. Mast cell sonicate increased fibroblast proliferation and contractile activity while decreasing collagen synthesis. Mast cell sonicate proliferating activities were found to be proteinase-sensitive with a molecular weight of more than 158 kd, of ∼40 kd, and of less than 10 kd. TGF-β and tryptase enhanced collagen synthesis; TNF-α and chymase decreased it. None of the selected mediators increased fibroblast proliferation. Conclusions: Myofibroblasts are the main connective tissue cells present in human peritoneal adhesions, and mast cells play a direct role in peritoneal adhesion formation.