Abstract. y-Methylaminobutyraldehyde (N-methylpyrroline) labeled with 14C was isolated from tobacco roots which had metabolized ornithine-2-'4C. It was labeled most strongly 4 hours after adding ornithine-2-14C to the root, also labeled by putrescine-1,4-1 C and methionine-4CH,, and observed in the root but not in the aerial portions of tobacco plants. y-Methylaminobutyraldehyde when added back to the root was an efficient precursor of nicotine. Identity of y-methylaminobutyraldehyde from tobacco roots was confirmed by comparison with the authentic compound.The existence of y-methylaminobutyraldehyde (N-methylpyrroline) in the biosynthetic pathway of nicotine indicates that the methyl group must be introduced before condensation of the pyridine moiety with the pyrrolidine moiety.It has been Nvell establishedl that the pyridine ring of nicotine is derived from nicotinic acid (4), and the pyrrolid,ine ring from ornithine (7,16), pltitrescine (17), glutamic acid, and proline (14).'rhe mechanism of the condensation of the 2 rings was dliscuissed by Bverrum and hi.s grouip (15), Leete et al. (18), Dawson (5), and Mothes and Schtitte (21). Byerruim and his co-workers (2,6) demonstrated that the methyl group is introdui,ced f,rom methionine anid labile C1-fragments. However, intermediattes between these precuirsors and nicotine anid the mechaniism of the reaction have remained largely lunexI)lored.Ladesic and Tso (13) and Schroter (22) have reported that nornicotine canl serve as a precursor of nicotine. However, ouir previous paper (11) demonstrated that while nicotine always occulrs in the pulre i-form in tobacco plants, nornicotine isola,te(l from tobacco roots is predominantly d-form. We a,lso reported that radioactive nicotine and nornicotine isolated from sterile root cuiltuires of Nicotiaina, ritstica fed DL-ornithine-2-1 4C d-iffered in the di,strilbution of radioactivity between C-2 and C-5 in the pyrrolidine ring, and that only small amounts of 15N were incorporated into nicotiie by administra,tion of 15N-labeled nornicotiine (20). These findings led to the inference that the biosynthesis of nicotine occurs through a routte(s) that does not involve nornicotine. Alworth and I Present address: