Tlr elements are a novel family of ~30 putative mobile genetic elements that are conﬁned to the germ line micronuclear genome in Tetrahymena thermophila. Thousands of diverse germ line-limited sequences, including the Tlr elements, are speciﬁcally eliminated from the differentiating somatic macronucleus. Macronucleusretained sequences ﬂanking deleted regions are known to contain cis-acting signals that delineate elimination boundaries. It is unclear whether sequences within deleted DNA also play a regulatory role in the elimination process. In the current study, an in vivo DNA rearrangement assay was used to identify internal sequences required in cis for the elimination of Tlr elements. Multiple, nonoverlapping regions from the ~23-kb Tlr elements were independently sufﬁcient to stimulate developmentally regulated DNA elimination when placed within the context of ﬂanking sequences from the most thoroughly characterized family member, Tlr1. Replacement of element DNA with macronuclear or foreign DNA abolished elimination activity. Thus, diverse sequences dispersed throughout Tlr DNA contain cis-acting signals that target these elements for programmed elimination. Surprisingly, Tlr DNA was also efﬁciently deleted when Tlr1 ﬂanking sequences were replaced with DNA from a region of the genome that is not normally associated with rearrangement, suggesting that speciﬁc ﬂanking sequences are not required for the elimination of Tlr element DNA.