Combinations of genes (aci and tcm, respectively) encoding subunits of polyketide synthases (PKSs) for actinorhodin (1) from Streptomyces coelicolor A3(2) and tetracenomycin (2) from Streptomyces glaucescens were functionally expressed in a recently developed Streptomyces host-vector system. Recombinant strains lacking any ketoreductase activity associated with the PKS produced novel polyketides, which were structurally characterized using NMR and high-resolution mass spectroscopy in combination with isotopic labeling experiments. As expected, the act and tcm PKSs produced completely unreduced polyketides with backbones derived from 8 and 10 acetate units, respectively (9 and 10). However, the regiospecificities of cyclizations in these two molecules differed from their reduced counterparts, and have not been previously observed in this class of bacterial polyketides. Taken together with earlier findings, our results provide evidence for yet another degree of freedom for the biosynthesis of novel polyketides through combinatorial expression of the act ketoreductase with PKSs of varying chain length specificities (including those that are not associated with any ketoreductase in nature). By comparing the structures and deduced backbones of the novel polyketides reported here with those of novel polyketides described earlier, a biosynthetic model is favored in which the regiospecificity of the first cyclization is controlled in part by the chain elongation enzymes themselves and is not influenced by the ketoreductase; however, the cyclase responsible for controlling the regiospecificity of the second cyclization can discriminate between polyketide backbones of different lengths as well as different degrees of reduction.