We investigated pulmonary endothelial function in vivo in 12- to 18-mo-old male Watanabe heritable hyperlipidemic (WHHL; n = 7) and age- and sex-matched New Zealand White (n = 8) rabbits. The animals were anesthetized and artificially ventilated, and the chest was opened and put in total heart bypass. The single-pass transpulmonary utilizations of the angiotensin-converting enzyme (ACE) substrate [³H]benzoyl-Phe-Ala-Pro (BPAP) and the 5'-nucleotidase (NCT) substrate [¹⁴C]AMP were estimated, and the first-order reaction parameter A_max/K_m, where A_max is the product of enzyme mass and the catalytic rate constant and K_m is the Michaelis-Menten constant, was calculated. BPAP transpulmonary utilization and A_max/K_m were reduced in WHHL (1.69 ± 0.16 vs. 2.9 ± 0.44 and 599 ± 69 vs. 987 ± 153 ml/min in WHHL and control rabbits, respectively; P < 0.05 for both). No differences were observed in the AMP parameters. BPAP K_m and A_max values were estimated separately under mixed-order reaction conditions. No differences in K_m values were found (9.79 ± 1 vs. 9.9 ± 1.31 µM), whereas WHHL rabbit A_max was significantly decreased (5.29 ± 0.88 vs. 7.93 ± 0.8 µmol/min in WHHL and control rabbits, respectively; P < 0.05). We conclude that the observed pulmonary endothelial ACE activity reduction in WHHL rabbits appears related to a decrease in enzyme mass rather than to alterations in enzyme affinity.