Nuclear matrix attachment regions (MARs) are thought to influence the expression of flanking genes. In this study, we investigated the activation of genes by tobacco MARs that had previously been identified in the 5' region of the basic class I chitinase gene, CHNS0. In transgenic tobacco cells, a construct consisted of the 35S promoter of cauliflower mosaic virus (CaMV) fused to a beta-glucuronidase gene (uidA) with 5' MAR elements was expressed at a 10-fold higher level than a similar construct without MAR sequences. However, expression of a similar construct with 3' MARs and of a construct with a truncated (-46) 35S minimal promoter and uidA with 5' MARs was not similarly enhanced, suggesting that MARs might act by increasing the activity of downstream enhancers. Deletion analysis of the MAR sequences revealed that the function of the MARs that increased the expression of the transgene was redundant. Moreover, assays of the transient expression of transgenes suggested that MAR elements might be involved in the structure and organization of chromatin. To examine the influence of MARs on chromatin structure, we investigated the effects of micrococcal nuclease (MNase) on the DNA in the reporter gene around the MARs. Analysis of the time-course of digestion of nuclei with MNase revealed that the 35S promoter region with 5' MARs was much more sensitive to MNase than the same region without MARs, suggesting that MARs might mediate the opening of chromatin in the region of a downstream promoter, with consequent enhancement of transcription.