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 α subunit (KSα) and ketoreductase, we conducted systematic localization experiments of the two proteins in Streptomyces coelicolor subproteomes. The results support the membrane location of KSα 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.