Vigilin, a multidomain hn-ribonucleo-K-homologous protein, is part of a ribonucleoprotein complex with cognate tRNA and is found in both the nucleus and the cytoplasm. In an approach to identify genomic regions involved in regulation of the chicken vigilin gene, we carried out transfection studies with a reporter gene in suitable chicken cells. After including a distantly positioned 5'-sequence in the construct, we observed a 10.5-fold increase in luciferase (EC 1. 13.12.7) expression compared with basal promoter activity. Accordingly, chromatin analysis of freshly isolated embryonic tendon fibroblasts with high levels of vigilin mRNA expression shows a DNase-I-hypersensitive site (DHS1) localized 2.2 kb upstream of the transcriptional start site. Similarly, phytohaemagglutinin-stimulated lymphocytes with a 4-fold elevated expression of vigilin mRNA compared with resting lymphocytes also exhibited this unique DHS, having switched from that found at 3.3 kb (DHS2) in resting lymphocytes. Furthermore, using gel-retardation experiments with DNA representing either DHS1 or DHS2, a specific interaction with chicken nuclear extracts was seen.