All of the binding sequences for MlcR, a transcriptional activator of ML-236B (compactin) biosynthetic genes in Penicillium citrinum, were identified by an in vitro gel-shift assay. All the identified sequences contain an asymmetric direct repeat comprised of conserved tetrad bases (A/T)CGG with a spacer sequence of high similarity; in particular, G at position 2 and T at position 3 in the spacer are well conserved. The first (A/T)CGG repeat was essential for MlcR-binding and MlcR could bind to this monomeric site, probably as a monomer. This binding feature might enable MlcR to tolerate the variation of the spacer length and compositions in vitro. From these data, we propose that the consensus binding motif for MlcR is an asymmetric direct repeat, 5′-(A/T)CGG-NGTN3-6-TCGG-3′.