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The identification of transcriptional regulatory modules within mammalian genomes is a prerequisite to understanding the mechanisms controlling regulated gene expression. While high-throughput microarray- and sequencing-based approaches have been used to map the genomic locations of sites of nuclease hypersensitivity or target DNA sequences bound by specific protein factors, the identification of regulatory elements using functional assays, which would provide important complementary data, has been relatively rare. Here we present a method that permits the functional identification of active transcriptional regulatory modules using a simple procedure for the isolation and analysis of DNA derived from nucleosome-free regions (NFRs), the 2% of the cellular genome that contains these elements. The more than 100 new active regulatory DNAs identified in this manner from F9 cells correspond to both promoter-proximal and distal elements, and display several features predicted for endogenous transcriptional regulators, including localization within DNase-accessible chromatin and CpG islands, and proximity to expressed genes. Furthermore, comparison with published ChIP-seq data of ES-cell chromatin shows that the functional elements we identified correspond with genomic regions enriched for H3K4me3, a histone modification associated with active transcriptional regulatory elements, and that the correspondence of H3K4me3 with our promoter-distal elements is largely ES-cell specific. The majority of the distal elements exhibit enhancer activity. Importantly, these functional DNA fragments are an average 149 bp in length, greatly facilitating future applications to identify transcription factor binding sites mediating their activity. Thus, this approach provides a tool for the high-resolution identification of the functional components of active promoters and enhancers.