Structurally diverse polyketides provide a rich reservoir of bioactive molecules. Actinorhodin, a model aromatic polyketide, is synthesized by minimal type II polyketide synthase and tailoring enzymes. The ActIII actinorhodin ketoreductase is a key tailoring enzyme in actinorhodin biosynthesis. With purified antibodies against actinorhodin polyketide synthase alpha subunit (KSalpha) and ketoreductase, we conducted systematic localization experiments of the two proteins in Streptomyces coelicolor subproteomes. The results support the membrane location of KSalpha and cell-wall location of ketoreductase. Considering previous evidence that some other tailoring enzymes of actinorhodin biosynthesis may be located outside the cytoplasm, a picture is emerging of an extensive role for extracellular biochemistry in the synthesis of type II polyketide antibiotic.