The distinction between a circulating renin-angiotensin system and a tissue renin-angiotensin system led us to determine tissue angiotensin converting enzyme (ACE) activity. This study establishes the experimental conditions for a good reproducibility of the fluorimetric assay of ACE and describes the use of [3H]ramiprilat to characterize ACE. Angiotensin converting enzyme activity was determined in rat lung, heart, aorta, and kidney (cortex and medulla) and in rabbit kidney (cortex, medulla, tubules, and glomeruli). ACE activity and [3H]ramiprilat binding does not increase in a linear fashion with the protein content of tissue extracts. Linearity limits varied from 1.0 to 2.0 mg of protein/ml (fluorimetry) and from 0.4 to 1.0 mg of protein/ml [( 3H]ramiprilat binding). Comparing ACE activity, measured by fluorimetry, with the amount of [3H]ramiprilat bound shows that the two techniques yield similar results.