Wnt signaling induces the nuclear accumulation of beta-catenin and transcription of specific target genes via the DNA-binding proteins, TCF/ Lef. While all known beta-catenin target genes encode proteins, genome-wide RNA profiling studies indicate that many transcripts do not have this capability. Transcription factor binding sites associated with these non-coding transcripts can be identified using unbiased techniques such as SACO (Serial Analysis of Chromatin Occupancy). We used this method to identify a beta-catenin-regulated antisense RNA expressed in HCT116 colorectal carcinoma cells, a cellular model of activated beta-catenin signaling. Genomic signature tags (GSTs) designating putative beta-catenin binding sites mapped to the 3' untranslated region (3' UTR) of the E2F4 gene. We showed that both beta-catenin and TCF4 bind to the E2F4 3' UTR site in vivo, inducing expression of an E2F4 antisense transcript. LiCl, which mimics Wnt signaling, also induced expression of the E2F4 antisense transcript and decreased E2F4 protein levels. This effect was blocked by a cDNA expressing the E2F4 3' UTR sense strand. The antisense-mediated decrease in E2F4 protein was reflected by reduced E2F4 association with specific target genes, including CCNA2, CDC2, PCNA, and Rad54. We propose that Wnt/ beta-catenin signaling may contribute to colorectal carcinogenesis by reducing the level of the E2F4 cell cycle repressor via an antisense mechanism.