The M1 muscarine agonist, 5-(2-ethyl-2H-tetrazol-5-yl)-1-methyl-1,2, 3,6-tetrahydropyridine (Lu 25-109), is extensively metabolized in mice, rats, dogs, and humans. The metabolite profile after an oral dose of [(14)C]Lu 25-109 was determined in plasma and in urine. Lu 25-109 was metabolized by N-demethylation (Lu 25-077), N-oxidation (Lu 32-181), and N-deethylation (Lu 31-126). In addition, combined N-demethylation and N-deethylation (Lu 31-190), and formation of a pyridine derivative took place (Lu 31-102). Lu 25-109 was also oxidized to pyridinium (Lu 29-297), 3-hydroxy-pyridinium (Lu 35-080), N-deethyl-2-pyridone (Lu 35-026), and a glucuronide of a 4, 6-dihydroxy-pyridinium ("m/z 398") compounds. A glucuronide of a dihydroxylated dihydro-pyridine compound ("m/z 400") was isolated from human urine, but not fully identified. In vitro studies were undertaken to elucidate the order of formation of the metabolites. In human plasma, the concentrations of Lu 25-109 and the pharmacologically active N-demethyl metabolite (Lu 25-077) were small compared with the N-oxide (Lu 32-181) and the N-deethyl-2-pyridone (Lu 35-026) at the first sample time (0.75 h). The N-deethyl metabolite (Lu 31-126) was the major component in human plasma between 3 and 10 h postdose. The major human metabolites in urine (Lu 32-181, Lu 35-026, and Lu 31-126) and the minor metabolites (Lu 25-077, Lu 35-080, Lu 31-190, and Lu 29-297) were all present in urine from rats, dogs, and mice, whereas m/z 398 was present in only mice and humans, and Lu 31-102 in only rats. The minor human metabolite m/z 400 was not detected in mice, rats, or dogs.