Tonin, a proteolytic enzyme isolated from the rat submandibular gland, can generate angiotensin II directly from angiotensinogen. To date a method for the measurement of tonin in plasma has not been available and the present paper describes a sensitive and specific excess antibody immunoassay for determination of tonin in tissue homogenates and plasma. Interference from immunologically cross-reacting proteins was evaluated and the assay was found to be specific for tonin. Tonin measured in various tissue homogenates was directly proportional to the amount of sample added, giving a linear dose-response curve. The slope of this curve was determined by the recovery of tonin, which was better than 55% for urine and all tissues tested. The highest concentration of tonin was seen in the submandibular and sublingual gland (69 and 0.7 microgram/mg protein, respectively). The parotid gland, the exorbital lacrimal gland, liver, kidney, pancreas, and lung contained only negligible amounts (less than 4 ng/mg protein). Tonin in plasma was bound to one major inhibitor with a molecular weight of about 650,000-750,000. A partial splitting of the tonin-inhibitor complex was obtained by preincubating plasma with guanidine, allowing tonin to be measured with a recovery of 38 +/- 13% (n = 16) and with a linear dose-response curve. The concentration of immunoreactive tonin in normal arterial plasma from adult male rats was 0.90 +/- 0.53 ng/ml (n = 16). The concentration decreased after removal of the submandibular glands and increased after sympathetic stimulation.