The Schizosaccharomyces pombe mfm1 gene is expressed in an M cell-specific fashion. This regulation requires two HMG-box proteins: the ubiquitous Ste11 transcription factor and the M cell-controlling protein Mat1-Mc. Here we report that the mfm1 promoter contains a single, weak Ste11-binding site (a so-called TR-box) that can confer M-specificity on a heterologous promoter when present in eight copies. In vitro, both Mat1-Mc and Ste11 can bind this box with approximately the same affinity. The Mat1-Mc protein caused a dramatic increase in the DNA-binding of Ste11 to this box, under conditions where we could not detect Mat1-Mc in the resulting protein-DNA complex. When we changed a single base in the mfm1 TR-box, such that it resembled those boxes found in ubiquitously expressed genes. Ste11 binding was enhanced, and in vivo the mfm1 gene also became expressed in P cells where Mat1-Mc is absent. These findings suggest that M-specificity results from Mat1-Mc-mediated Ste11 binding to weak TR-boxes. We have also defined a novel motif (termed M-box), adjacent to the mfm1 TR-box, to which Mat1-Mc binds strongly. A DNA fragment containing both the TR- and the M-box allowed the formation of a complex containing both Ste11 and Mat1-Mc. A single copy of this fragment was sufficient to activate a heterologous promoter in an M-specific fashion, suggesting that these two boxes act in a synergistic manner.