The mode of incorporation of 2H-labelled putrescines into the rosmarinecine (14) portion of the pyrrolizidine alkaloid rosmarinine (15) in Senecio pleistocephalus plants has been established by 2H n.m.r. spectroscopy. The use of [1,1,4,4-²H₄] putrescine (16) dihydrochloride produced rosmarinine (18) with ²H labels mainly at C-3α, C-3β, and C-9pro-S. Rosmarinine (22) derived from [1,1-²H₂] putrescine (19) dihydrochloride showed the same ²H n.m.r. spectroscopic signals plus others for C-5α, C-5β, and C-8. These labelling patterns are interpreted by the intermediacy of homospermidine (6) in the biosynthetic pathway and by the existence of ²H isotope effects. Feeding experiments with (R)-[1-²H]- (23) and (S)-[1-²H] putrescine (26) dihydrochloride gave a sample of rosmarinine (25) with ²H present at C-3β, C-5α, C-8α, and C-9pro-S from the former precursor, and a sample (28) with ²H present at C-3α and C-5β from the latter precursor. These patterns are consistent with oxidation of three of the four amino groups involved in the conversion of two molecules of putrescine into 1β-formyl-Δα-pyrrolizidine (12) with stereospecific loss of the pro-S hydrogens. Reduction of the 1-formylpyrrolizidine (12) to isoretronecanol (13) takes place by attack of a hydride donor on the C-re face of the aldehyde group. Use of [2,2,3,3-²H₄] putrescine (29) dihydrochloride gave rosmarinine (30) labelled with ²H at C-1α, C-2β, C-6α, C-6β, and C-7α. Feeding experiments with (R)-[2-²H]- (31) and (S)-[2-²H] -putrescine (33) dihydrochloride gave rosmarinine (32) labelled with ²H at C-2β and C-6α from the former, and rosmarinine (34) with ²H present at C-1α, C-6β, and C-7α from the latter precursor. Formation of the pyrrolizidine ring involves stereospecific removal of the pro-R hydrogen and retention of the pro-S hydrogen on the carbon destined to become C-1 of rosmarinecine, and hydroxylation at C-2 and C-7 of isoretronecanol (13) therefore proceeds with retention of configuration to form rosmarinecine (14).