Human CD2 locus control region (LCR) sequences are shown here to be essential for establishing an open chromatin configuration. Transgenic mice carrying an hCD2 mini-gene attached only to the 3' CD2 transcriptional enhancer exhibited variegated expression when the transgene integrated in the centromere. In contrast, mice carrying a transgene with additional 3' sequences showed no variegation even when the latter integrated in centromeric positions. This result suggests that LCRs operate by ensuring an open chromatin configuration and that a short region, with no enhancer activity, functions in the establishment, maintenance, or both of an open chromatin domain.