Recombinant beta-amyrin synthase from Pisum sativum converted 24,30-bisnor-2,3-oxidosqualene into a 3:1:0.2 mixture of 29,30-bisnor-beta-amyrin, 29,30-bisnorgermanicol, and 29,30-bisnor-delta-amyrin. Further, enzyme reactions with [23-13C]- and [23,23-2H]-labeled isotopomers demonstrated that the cyclization did not proceed through formation of a lupanyl primary cation with a five-membered E-ring, but an electrophilic addition of the tetracyclic C-18 cation on to the terminal double bond directly generated a thermodynamically favored pentacyclic secondary cation with a less-strained six-membered E-ring. Interestingly, the formation of the three regioisomers suggested that the absence of the terminal methyl groups resulted in a structural perturbation in the folding conformation of the E-ring of the oleanyl cation at the active site of the enzyme.