<jats:p> The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4 <jats:italic>R</jats:italic> ,5 <jats:italic>R</jats:italic> )-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4 <jats:italic>R</jats:italic> ,5 <jats:italic>R</jats:italic> )-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by <jats:italic>fkbO</jats:italic> in the FK506 and FK520 biosynthetic gene clusters, and by <jats:italic>rapK</jats:italic> in the rapamycin gene cluster of <jats:italic>Streptomyces hygroscopicus</jats:italic> . Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using <jats:italic>fkbO</jats:italic> from either the FK506 or the FK520 gene cluster of a strain of <jats:italic>S. hygroscopicus</jats:italic> specifically deleted in <jats:italic>rapK</jats:italic> (BIOT-4010) restored rapamycin production, as did supplementation with (4 <jats:italic>R</jats:italic> ,5 <jats:italic>R</jats:italic> )-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the <jats:italic>rapK</jats:italic> homolog <jats:italic>hyg5</jats:italic> as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the <jats:italic>bra8</jats:italic> gene from the pathway to the terpenoid natural product brasilicardin. Expression of either <jats:italic>hyg5</jats:italic> or <jats:italic>bra8</jats:italic> in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.