Enzymatic activity of tonin-alpha 1-macroglobulin complex was studied in vitro and in vivo, using an immunoimmobilization technique. Tonin-alpha 1-macroglobulin complex, which was immunologically immobilized by anti-alpha 1-macroglobulin antibody covalently coupled to agarose gels, could quantitatively hydrolyze angiotensin I and synthetic tridecapeptide renin substrate to form angiotensin II. However, the solid-phase antibody-bound tonin-alpha 1-macroglobulin complex could not hydrolyze the plasma protein renin substrate. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, inhibited both free tonin and the solid-phase antibody-bound tonin-alpha 1-macroglobulin complex. The hydrolytic activity of the solid-phase antibody-bound tonin-alpha 1-macroglobulin complex against angiotensin I was not inhibited by soybean trypsin inhibitor (molecular weight, 23,000), a potent inhibitor of free tonin. Taken together, these results suggest that tonin bound to alpha 1-macroglobulin keeps the active site intact and that inhibition of the enzyme activity is due to a steric hindrance. When 500 microliter of tonin was administered intravenously to rats, the immunoimmobilization method was used to show that the tonin-alpha 1-macroglobulin complex in the plasma formed angiotensin II. Thus, the tonin-alpha 1-macroglobulin complex in the plasma may be linked to some forms of hypertension through angiotensin II formation.