The biosynthesis of the tropane alkaloids in transformed root cultures of Datura stramonium has been studied using sodium [1,2-13C2]acetate, (R,S)-[2,3-13C2]-1-(1-methyl-2-pyrrolidinyl)propan-2-one {(R,S)-[2′,3′-13C2] hygrine}, ethyl (R,S)-[1,2-13C2,2-14C]-2-(1-methyl-2-pyrrolidinyl)acetate, and ethyl (R,S)-[2,3-13C2,3-14C]-4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate. The incorporation of (R,S)-[2′,3′-13C2]hygrine into cuscohygrine and several other condensation products was high (15-40% specific incorporation), but label was not recovered in either tropine or tropine esters (hyoscyamine; 0.0 ( 0.5% specific incorporation). None of the recovered alkaloids was labeled when ethyl (R,S)-[1,2-13C2,2-14C]-2-(1-methyl-2-pyrrolidinyl)acetate was fed to the cultures. In contrast, sodium [1,2- 13C2]acetate and ethyl (R,S)-[2,3-13C2,3-14C]-4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate were incorporated into hyoscyamine (9 and 2% specific incorporation, respectively) and a number of other tropane alkaloids (up to 12% specific incorporation). These data provide further evidence that hygrine is not a direct precursor of tropane alkaloids. 13C-Label from acetate was incorporated symmetrically into the C-2 and C-4 positions of (-)-hyoscyamine. The evidence supports a pathway in which acetoacetate reacts via its C-4 position with N-methyl-∆1-pyrrolinium salt to give 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate. This intermediate favors cyclization to give 2-carboxytropinone, tropinone being formed by decarboxylation.