Barbamide is a mixed polypeptide-polyketide natural product that contains an unusual trichloromethyl group. The origin of the trichloromethyl group was previously shown to be through chlorination of the pro-R methyl group of L-leucine. Trichloroleucine is subsequently decarboxylated and oxidized to trichloroisovaleric acid and then extended with an acetate unit to form the initial seven carbons of barbamide. In this study we used a combination of biosynthetic feeding experiments and enzymatic analysis to characterize the initial steps required for formation of trichloroleucine and its chain-shortened product, trichloroisovaleric acid. Results from isotope-labeled feeding experiments showed that both dichloroleucine and trichloroleucine are readily incorporated into barbamide; however, monochloroleucine is not. This suggests that halogenation of the pro-R methyl group of leucine occurs as two discrete reactions, with the first involving incorporation of at least two halogen atoms and the second converting dichloroleucine to trichloroleucine. Additionally, the initial tandem dichlorination must occur before substrate can be further processed by the remainingbar pathway enzymes. In vitro analysis of the first five open reading frames (ORFs; barA, barB1, barB2, bar C, barD) of the barbamide gene cluster has yielded new insights into the processing of leucine to form the trichloroisovaleryl-derived unit in the final product.