(1R)-1-3H-labeled and (1S)-1-3H-labeled geranyl pyrophosphate and neryl pyrophosphate were prepared from the corresponding 1-3H-labeled aldehydes by a combination of enzymatic and synthetic procedures. Following admixture with the corresponding 2-14C-labeled internal standard, each substrate was converted to (+)-bornyl pyrophosphate and (-)-bornyl pyrophosphate by cell-free enzyme preparations from sage (Salvia officinalis) and tansy (Tanacetum vulgare), respectively. Each pyrophosphate ester was hydrolyzed, and the resulting borneol was oxidized to camphor. The stereochemistry of labeling at C-3 of the derived ketone was determined by base-catalyzed exchange, taking advantage of the known selective exchange of the exo-alpha-protons. By comparison of such exchange rates to those of product generated from (1RS)-2-14C,1-3H2-labeled substrate, it was demonstrated that geranyl pyrophosphate was cyclized to bornyl pyrophosphate with net retention of configuration at C-1 of the acyclic precursor, whereas neryl pyrophosphate was cyclized to product with inversion of configuration at C-1. The observed stereochemistry is consistent with a reaction mechanism whereby geranyl pyrophosphate is first stereospecifically isomerized to linalyl pyrophosphate which, following rotation about C-2-C-3 to the cisoid conformer, cyclizes from the anti-endo configuration. Neryl pyrophosphate cyclizes either directly or via the linalyl intermediate without the attendant rotation.