Mutations in a number of genes responsible for the maintenance of transposon silencing have been reported. However, the initiation of epigenetic silencing of transposable elements is poorly characterized. Here, we report the identification of a single dominant locus, Mu killer (Muk), that acts to silence MuDR, the autonomous regulatory transposon of the Mutator family of transposable elements in maize. Muk results in the methylation of MuDR TIRs and is competent to silence one or several active MuDR elements. Silencing by Muk is not dependent on the position of the MuDR element and occurs gradually during plant development. Transcript levels of the MuDR transposase, mudrA, decrease substantially when Muk is present. The other transcript encoded by MuDR, mudrB, also fails to accumulate in the poly(A) RNA fraction when MuDR and Muk are combined. Additionally, plants undergoing MuDR silencing produce small, mudrA-homologous approximately 26-nt RNAs, suggesting a role for RNA-directed DNA methylation in MuDR silencing. MuDR elements silenced by Muk remain silenced even in plants that do not inherit Muk, suggesting that Muk is required for the initiation of MuDR silencing but not for its maintenance.