We have examined the chromatin structure of a 28-kb chromosomal region containing the gene (CaBP9k) encoding calbindin-D9k in different rat tissues. DNase I-hypersensitive sites (HSs) were probed with DNase I using an indirect end-labeling technique. Duodenal chromatin, where the gene is strongly expressed, contained one major HS (HS4) and three minor HSs (HS2, HS3 and HS5) near the promoter region. The HS4 was mapped just upstream from the promoter region and had the characteristics of a tissue-specific HS. The HS5 was located at the transcription start point, it included the TATA box and its presence was correlated with a promoter function. The duodenal chromatin contained two additional HSs, a major HS (HS1) located approx. 3.5 kb upstream from the cap site and a minor HS (HS6) in the second intron at +0.5 kb. Despite the marked effect of 1,25-dihydroxyvitamin D3 (DHD3) on the CaBP9k mRNA level, the pattern of HSs in duodenal chromatin was unchanged after stimulation with DHD3. The liver chromatin contained one major HS (HS1) identical to the duodenum HS1 (although this tissue does not express CaBP9k). It also contained a liver-specific HS (HS0) 0.1 kb upstream from HS1. The interaction of HS0 with HS1 could explain the absence of CaBP9k expression in the liver. Thus, specific sets of HSs are associated with various functional states of CaBP9k in a tissue-specific manner.