Human alpha thrombin acts as a mitogen for cultures of resting chick embryo fibroblasts (CEF) in serum free medium. The use of 125I-labeled thrombin shows that thrombin specifically binds to CEF and that after a lag of approximately 30 to 60 minutes it can not be removed by subsequent exposure to trypsin. The entry of 125I thrombin into the trypsin-insensitive domain is not inhibited to any great extent by excess unlabelled thrombin. The cell-associated thrombin retains its native molecular weight and its catalytic activity toward synthetic amide substrates. It appears to be located in the crude nuclear fraction of homogenized CEF cells. The association of thrombin with CEF is specific, since the non-mitogenic serine protease chymotrypsin is internalized to a much lesser extent than thrombin. The data are discussed in terms of a possible intracellular site for thrombin's mitogenic action.