Abstract Glutamate, a proposed precursor of the pyrrolidine ring of nicotine, was isolated and degraded after metabolism of 14CO2 for 6 hours by Nicotiana rustica and Nicotiana glutinosa and after metabolism of acetate-2-14C for 2 hours by N. rustica. The 14C distribution in glutamate from N. glutinosa after 6 hours of 14CO2 metabolism was 20.2, 14.3, 13.9, 24.4, and 26.6%, in C-1 through C-5, respectively. The 14C distribution in nicotine under the same conditions was pyridine ring, 77.8%; C-2', 4.3%; C-3', 4.4%; C-4', 4.7%; C-5', 4.1%; and methyl carbon, 5.0%. The labeling pattern of the pyrrolidine ring is in agreement with the symmetrical intermediate pathway proposed for the formation of the pyrrolidine ring of nicotine from glutamate. Similar results were obtained after 6 hours of 14CO2 incorporation into nicotine by N. rustica. After 3 hours of 14CO2 metabolism by N. glutinosa, 90.4% of the radioactivity was in the pyridine ring, 5.4% in the methyl group, and the remaining radioactivity was distributed equally among the carbon atoms of the pyrrolidine ring. Acetate-2-14C labeled glutamate in N. rustica as would be anticipated if acetate were metabolized via the tricarboxylic acid cycle: 1.5%, 5.4%, 4.6%, 81.0%, 2.8% in C-1 through C-5, respectively. The distribution of 14C in nicotine was: pyridine ring, 64.4%; C-2', 2.6%; C-3', 16.4%; C-4', 15.6%; C-5', 1.8%; and methyl carbon, 1.6%. These results support the symmetrical intermediate hypothesis for the formation of the pyrrolidine ring of nicotine proposed on the basis of previous precursor experiments.