The biosynthesis of the tropane alkaloids in Datura innoxia was studied using (R,S)-[2’,3’-C-13(2)]hygrine and ethyl (R,S)-[2,3-C-13(2),3-C-14]-4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate. (R,S)-[2’,3’-C-13(2)]Hygrine was synthesized from ethyl [3,4-C-13(2)]acetoacetate. Administration of (R,S)-[2’,3’-C-13(2)]hygrine to D. innoxia resulted in a very low incorporation of C-13 into (-)-scopolamine and (-)-hyoscyamine. Ethyl (R,S)-[2,3-C-13(2),3-C-14]-4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate was synthesized from potassium [C-13,C-14]cyanide and diethyl [2-C-13]malonate. Administration of ethyl (R,S)-[2,3-C-13(2),3-C-14]-4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate to D. innoxia resulted in a high incorporation of both C-14 (1.73% specific incorporation) and C-13 (1.78% specific incorporation) into (-)-scopolamine. This provides direct evidence for an alternative biosynthetic pathway for the tropane alkaloids in D. innoxia.