An increase in compactin (ML-236B) production was achieved by introducing a whole compactin biosynthetic gene cluster or the regulatory gene mlcR into compactin high-producing Penicillium citrinum. In the previous report, we introduced mlcR encoding the positive regulator of compactin biosynthetic genes into compactin high-producing strain no. 41520, and most of the transformants produced higher amounts of compactin. Here, we characterize one of the resulting high producers (strain TIR-35, which produced 50% more compactin) and reveal that TIR-35 contained five copies of mlcR and that early, enhanced expression of mlcR caused compactin overproduction. Similarly, the introduction of mlcR into strain T48.19, which was created previously from strain no. 41520 by introducing a partial compactin biosynthetic gene cluster, enhanced compactin production further. Our results indicated that genetic engineering is an effective tool to improve compactin production, even in compactin high producers.