To study the role of matrix-associated regions (MARs) in establishing independent chromatin domains in plants, two transgenes were cloned between chicken lysozyme A elements. These transgenes were the neomycin phosphotransferase (NPTII) gene under control of the nopaline synthase (nos) promoter and the [beta]-glucuronidase (GUS) gene controlled by the double cauliflower mosaic virus (dCaMV) 35S promoter. The A elements are supposed to establish an artificial chromatin domain upon integration into the plant DNA, resulting in an independent unit of transcriptional regulation. Such a domain is thought to be characterized by a correlated and position-independent, hence copy number-dependent, expression of the genes within the domain. The presence of MARs resulted in a higher relative transformation efficiency, demonstrating MAR influence on NPTII gene expression. However, variation in NPTII gene expression was not significantly reduced. The selection bias for NPTII gene expression during transformation could not fully account for the lack of reduction in variation of NPTII gene expression. Topological interactions between the promoter and A element may interfere with the A element as a domain boundary. In contrast, the GUS gene on the same putative chromatin domain showed a highly significant reduction in variation of gene expression, as expected from previous results. Surprisingly, no copy number-dependent GUS gene expression was observed: all plants showed approximately the same GUS activity. We concluded, therefore, that dCaMV 35S-GUS gene expression in mature tobacco plants is regulated by some form of dosage compensation.