A 75 kb gene cluster in Pseudomonasfl uorescens NCIMB 10586 encodesa modular polyketide synthase responsible for biosynthesis of the antibiotic mupirocin. All seven PKS modules lack intrinsic acyl transferase (AT) domains. Two AT domains within the multifunctional polypeptide MmpC are thought to operate in trans. An unusually large tailoring region encodes 26 discrete proteins that modify the polyketide/fatty acid backbone, in addition to providing resistance and regulatory functions. This study investigated the functions of four tailoring region genes. MupJ, MupK and mAcpC were revealed to be involved in insertion of a β-methyl group, while MupN was shown to have phosphopantetheinyl transferase activity. Five tailoring acyl carrier proteins (mAcps) were found to be specific and non-interchangeable, and the influence of C-termini on mAcp-specificity was investigated. Attempts to overexpress mAcps in P. fluorescens revealed that a broad-host-range IncP-1 vector is unstablein this host,p romptingt he constructiono f a new IncP-9v ector for use in P. fluorescens. The importance of the two ATs and the third domain of MmpC were demonstrated by mutagenesis and complementation. Overexpression of AT domains did not increase mupirocin production but specific heterologous trans-AT domains could malonylate the mupirocin PKS.