The location of regions that regulate transcription of the murineEmilin1gene was investigated in a DNA fragment of 16.8 kb, including the entire gene and about 8.7 and 0.6 kb of 5′- and 3′-flanking sequences, respectively. The 8.7-kb segment contains the 5′-end of the putative2310015E02Rikgene and the sequence that separates it fromEmilin1, whereas the 0.6-kb fragment covers the region betweenEmilin1andKetohexokinasegenes. Sequence comparison between species identified several conserved regions in the 5′-flanking sequence. Most of them contained chromatin DNase I-hypersensitive sites, which were located at about –950 (HS1), –3100 (HS2), –4750 (HS3), and –5150 (HS4) in cells expressingEmilin1mRNA.Emilin1transcription initiates at multiple sites, the major of which correspond to two Initiator sequences. Promoter assays suggest that core promoter activity was mainly dependent on Initiator1 and on Sp1-binding sites close to the Initiators. Moreover, one important regulatory region was contained between –1 and –169 bp and a second one between –630 bp and –1.1 kb. The latter harbors a putative binding site for transcription factor AP1 matching the location of HS1. The function of different regions was studied by expressinglacZconstructs in transgenic mice. The results show that the 16.8-kb segment contains regulatory sequences driving high level transcription in all the tissues whereEmilin1is expressed. Moreover, the data suggest that transcription in different tissues is achieved through combinatorial cooperation between various regions, rather than being dependent on a singlecis-activating region specific for each tissue.