We developed an assay for aminopeptidase (\mathrm{N}) (AmN) in which substrate, Arg-Phe- (\left[{ }^{3} \mathrm{H}\right]) anilide ((24.9 \mathrm{Ci} /) mmol), can be used at concentrations ( (1-200 \mathrm{nM}) ) well below (K_{m}(12 \mu \mathrm{M})) and at or below enzyme concentration ([E]). Such reaction conditions simulate those in vivo where peptide hormones in picomolar concentrations (\left(\ll K_{m}\right.) ) are degraded by nano- or micromolar concentrations of enzyme. The Arg-Phe- (\left[{ }^{3} \mathrm{H}\right]) anilide: (\mathrm{AmN}) reaction obeyed first-order enzyme kinetics when human serum, human seminal plasma, guinea pig serum, or homogeneous porcine kidney (A \mathrm{mN}) was used as enzyme source and substrate was within the concentration range of 1-200 nM. For porcine (\mathrm{AmN}, \boldsymbol{k}{\mathrm{cat}} / \boldsymbol{K}{m}) was 1.47 (\times 10^{9} \mathrm{M}^{-1} \min ^{-1}, k_{\text {cat }} 17,640 \mathrm{~min}^{-1}). Human serum AmN was in a concentration (about (4.6 \mathrm{nM}) ) in great excess over those reported for substrates such as angiotensin III. Several advantages accrue under conditions of first-order enzyme kinetics: (1) (V_{\max } / K_{m}) is measured directly. (2) When (\boldsymbol{k}{\text {cat }} / K{m}) is known, [E] can be computed in (\mathrm{mol} /) liter. (3) (\mathrm{IC}{50}) values for alternative substrates can be taken as (K{m}) values. (4) (\mathrm{IC}{\text {bะพ }}) values for inhibitors are (K{i}) values when (K_{i} \gg[E]). Arg-Phe- (\left[{ }^{3} H\right]) anilide can be used to measure AmN activity in the presence of chromophores and fluorophores that interfere with photometric and fluorometric assays. We have confirmed that alleged substrates such as angiotensin III and MetLys- and Lys-bradykinin are bound by AmN with high affinities ( (\boldsymbol{K}_{m}) values, 5.7, 9.1, and (14.3 \mu \mathrm{M}) ). Bovine pulmonary artery endothelial cell cultures were found to possess AmN-like activity. Given the wide distribution of AmN on cell surfaces and its high affinities for the peptide hormones tested, it appears increasingly plausible that AmN functions in vivo as a peptide hormone degrading enzyme. (C) 1993 Academic Press, Inc.