Glucagon has a short plasma t_1/2 in vivo, with renal extraction playing a major role in its elimination. Glucagon is degraded by neutral endopeptidase (NEP) 24.11 in vitro, but the physiological relevance of NEP 24.11 in glucagon metabolism is unknown. Therefore, the influence of candoxatril, a selective NEP inhibitor, on plasma levels of endogenous and exogenous glucagon was examined in anesthetized pigs. Candoxatril increased endogenous glucagon concentrations, from 6.3±2.5 to 20.7±6.3 pmol/l (C-terminal RIA, p<0.05). During glucagon infusion, candoxatril increased the t_1/2 determined by C-RIA (from 3.0±0.5 to 17.0±2.5 min, p<0.005) and mid-region (M) RIA (2.8±0.5 to 17.0±3.0 min, p<0.01) and reduced metabolic clearance rates (MCR, 19.1±3.2 to 9.4±2.0 ml/kg/min, p<0.02, C-RIA; 19.2±4.8 to 9.0±2.3 ml/kg/min/, p<0.05, M-RIA). However, neither t_1/2 nor MCR determined by N-RIA were significantly affected (t_1/2, 2.7±0.4 to 4.5±1.6 min; MCR, 30.3±6.4 to 28.5±9.0 ml/kg/min), suggesting that candoxatril had no effect on N-terminal degradation, but leads to the accumulation of N-terminally truncated forms of glucagon. Determination of arterio-venous glucagon concentration differences revealed that renal glucagon extraction was reduced (but not eliminated) by candoxatril (from 40.4±3.8 to 18.6±4.1%, p<0.02, C-RIA; 29.2±3.1 to 14.7±2.2%, p<0.02, M-RIA; 26.5±4.0 to 19.7±3.5%, p<0.06, N-RIA). Femoral extraction was reduced by candoxatril when determined by C-RIA (from 22.7±2.4 to 8.0±5.1%, p<0.05), but not changed significantly when determined using M- or N-RIAs (10.0±2.8 to 4.7±3.7%, M-RIA; 10.5±2.5 to 7.8±4.2%, N-RIA). This study provides evidence that NEP 24.11 is an important mediator of the degradation of both endogenous and exogenous glucagon in vivo