ABSTRACT We studied the silencing of the cryptic mating-type loci HML α and HMR a in the budding yeast Kluyveromyces lactis . A 102-bp minimal silencer fragment was defined that was both necessary and sufficient for silencing of HML α. Mutagenesis of the silencer revealed three distinct regions (A, B, and C) that were important for silencing. Recombinant K. lactis ribosomal DNA enhancer binding protein 1 (Reb1p) could bind the silencer in vitro, and point mutations in the B box abolished both Reb1p binding and silencer function. Furthermore, strains carrying temperature-sensitive alleles of the REB1 gene derepressed the transcription of the HML α 1 gene at the nonpermissive temperature. A functional silencer element from the K. lactis cryptic HMR a locus was also identified, which contained both Reb1p binding sites and A boxes, strongly suggesting a general role for these sequences in K. lactis silencing. Our data indicate that different proteins bind to Kluyveromyces silencers than to Saccharomyces silencers. We suggest that the evolution of silencers is rapid in budding yeasts and discuss the similarities and differences between silencers in Saccharomyces and Kluyveromyces .