The substrate specificity of tonin from rat submaxillary gland was examined with a series of synthetic peptides encompassing the C-terminus of the decapeptide substrate angiotensin I. In contrast to angiotensin I-converting enzyme from plasma or lung, only angiotensin I, (des-Asp1)-angiotensin I, and (des-Asp1, des-Arg2)-angiotensin I are substrates of tonin with Km values of 34.5 muM, 39.3 muM, and 54.4 muM, respectively, while the shorter C-terminal peptides are not hydrolyzed. Thus, the N-terminal sequence extending from position 1 to 3 is the enzymatic binding site for tonin. Turnover numbers of 33.4 sec-1, 42.8 sec-1, and 6.5 sec-1 are observed for the hydrolysis of angiotensin I, (des-Asp1)-angiotensin I, and (des-Asp1, des-Arg2)-angiotensin I, respectively. The relative percentage rates of hydrolysis (proportional to V/Km) at low substrate concentrations ([S] less than less than Km) are almost identical for (des-Asp1)-angiotensin I, angiotensin I, and the tetradecapeptide substrate, indicating that these three peptides are equally good substrates at low physiological concentrations. The observed high specificity of the enzyme lends support to the possible important role of tonin for local conversion in tissue. The conversion of (des-Asp1)-angiotensin I to (des-Asp1)-angiotensin II (angiotensin III) is of particular interest in relation to the recently suggested, potential role of the latter peptide in aldosterone release.