TATA-box-binding protein (TBP)-related factor (Trf)2 is a member of the family of TBP-related factors present in metazoan organisms. In Drosophila, Trf2 immunoprecipitates with the nucleosome remodeling factor (NURF) chromatin remodeling complex and the DNA replication element (DRE)-binding factor DREF. When it forms a complex with DREF, Trf2 activates transcription from the DRE-binding sites of the proliferating cell nuclear antigen (PCNA) gene. Despite these observations at the molecular level, no mutations in the trf2 locus have been found in Drosophila. Here, we identify two P-element insertion alleles, PL28 and GS7403, as hypomorphic mutants with a decreased expression level of trf2. Pupae of these mutant alleles show failure in anterior spiracle eversion, a hallmark of mutations in the loci associated with ecdysteroid signaling.