The tetracenomycin polyketide synthase (TCM PKS), a type II complex, produces TCM F2, a precursor of TCM C in Streptomyces glaucescens, and consists of at least the TcmK, -L, -M, and -N proteins. The TcmK/TcmL ketosynthase subunits were purified from overexpression of their genes in Streptomyces lividans. TcmK (calculated molecular mass 45 kd) and TcmL (calculated molecular mass 42 kd) function as a heterodimeric alphabeta complex based on observing that only the purified complex complemented TCM PKS activity in protein extracts made from strains bearing tcmK or tcmL deletion mutants to make TCM F2 in vitro, and that the molecular mass of the purified complex was 90 kd as estimated by gel filtration chromatography. The TCM PKS activity was reconstituted with purified protein components, indicating that the minimal set of proteins required to make TCM F2 included the ketosynthase complex (TcmKL), an acyl carrier protein (TcmM), a malonyl CoA:ACP acyltransferase (MAT), and a cyclase (TcmN). The MAT was required to catalyze the transacylation between malonyl-CoA and TcmM, although a relatively slow spontaneous transacylation also occurred in a reaction without the MAT. Acetyl-CoA, the proposed starter unit for the TCM PKS, was not required for the production of TCM F2 in vitro, although it could be incorporated into this polyketide to a small extent. TcmJ, a PKS protein without a known function, greatly increased the production of TCM F2 but could not replace TcmN as a cyclase in the reconstituted system.