6-Deoxyerythronolide B synthase (DEBS), the multifunctional enzyme responsible for the biosynthesis of the macrolide aglycon of the antibiotic erythromycin, is an excellent model system for studying the properties of modular polyketide synthases. In these studies, we analyzed the substrate specificity of selected individual modules of DEBS. Unexpectedly, we observed (i) a high degree of similarity in the specificity of all modules tested, despite the diverse structural features of their natural substrates, and (ii) a distinct preference by all modules for syn diketides over anti diketides. The implications of these results are analyzed from an evolutionary and a protein engineering perspective.