The mechanism by which the fatty acid acetylenase of Crepis alpina catalyzes crepenynic acid ((9Z)-octadeca-9-en-12-ynoic acid) production from linoleic acid has been probed through the use of kinetic isotope effect (KIE) measurements. This was accomplished by incubating appropriate mixtures of regiospecifically deuterated isotopomers with a strain of Saccharomyces cerevisiae expressing a functional acetylenase. LC/MS analysis of crepenynic acid obtained in these experiments showed that the oxidation of linoleate occurs in two discrete steps, since the cleavage of the C12-H bond is very sensitive to isotopic substitution (k(H)/k(D) = 14.6 +/- 3.0) while a minimal isotope effect (k(H)/k(D) = 1.25 +/- 0.08) was observed for the C13-H bond breaking step. These data suggest that crepenynic acid is produced via initial H-atom abstraction at C12 of a linoleoyl substrate. The relationship between the mechanism of enzymatic acetylenation and epoxidation is discussed.