The utrophin gene codes for a large cytoskeletal protein closely related to dystrophin. Its transcription is driven by a TATA-less promoter. Here we analyzed 40 kilobases of the 5' end region of the utrophin gene searching for new utrophin regulatory elements in muscle cells. By transient transfection of utrophin genomic fragments in front of a reporter gene, we identified a new enhancer that maps downstream of the transcription start site within the second intron and co-localizes with a DNase I-hypersensitive site. By deletion analysis it was mapped to a sequence of 128 base pairs that retains the whole activity. Linker scanning mutagenesis showed that most of the enhancer sequence is essential for its transcriptional activity. Binding analysis with nuclear cell extracts demonstrated that the enhancer regulatory elements, identified by mutagenesis, are protected from DNase I digestion. Because utrophin can functionally substitute dystrophin, the identification and characterization of new regulatory elements provide new targets for possible therapies of Duchenne muscular dystrophy aiming at the up-regulation of the utrophin expression in muscle cells.