Rat connective tissue mast cells are known to store significant amounts of mast cell protease I (RMCP I), which suppresses normal cell growth and mediates cytotoxicity against tumor cell lines, including the fibrosarcoma cell line FL. To better define its effects on FL cells, RMCP I was added to FL cultures for 30 min. Analysis of de novo nuclear protein synthesis revealed that RMCP I suppressed the expression of three proteins (41, 46, and 69 kD) and enhanced the expression of two other proteins (25 and 32 kD). Treatment of FL cells with diisopropylfluorophosphate (DFP)-inactivated RMCP I proved that these effects were largely independent of the protease catalytic site. Western blot hybridization, using a monoclonal antibody to phosphotyrosine-containing proteins, revealed that RMCP I inhibited phosphorylation of a nuclear and a cytoplasmic 81-kD tyrosylprotein. Inhibition of nuclear tyrosine kinase activity by RMCP I appeared to be catalytic site dependent, whereas cytoplasmic tyrosine kinase inhibition was independent of RMCP I proteolytic activity. Biotinylated RMCP I was used to identify potential surface-binding proteins. Three specific binding complexes (130, 150, and 210 kD) were detected. The binding of biotinylated RMCP I to these surface proteins was inhibited by excess unlabeled RMCP I, but not by trypsin or chymotrypsin. We speculate that the binding proteins may be critical in initiating RMCP I-induced metabolic changes on FL cells. The ability of RMCP I to alter the metabolism of cells suggests that it may have an important role in regulating their functions.