The regulation of major histocompatibility complex (MHC) class II genes expression, which can be constitutive, inducible or both, is a crucial aspect of the control of an immune response. It involves binding of various regulatory factors to cis-acting sequences of MHC class II promoters. Antisense oligonucleotides specific for RFX-1, a regulatory factor binding to the functionally essential X box motive of MHC class II promoters, were designed to study the role of RFX-1 in the various modes of MHC class II regulation and explore the possibility of experimentally modulating the level of expression of MHC class II genes by transcriptional intervention. RFX-1 antisense oligonucleotides were first tested in cell-free translation, selected for an inhibitory effect on RFX-1 in vitro translation and then assayed in cell cultures for an effect on human histocompatibility leukocyte antigen (HLA) class II expression. We show that an RFX-1 specific antisense oligonucleotide drastically inhibits induction of HLA-DR,-DQ, and -DP molecules by interferon gamma in monocytic cells. Unexpectedly, the same agent has no effect on the constitutive expression of the same genes either in these cells or in B lymphocytes, indicating an uncoupling of the constitutive and inducible modes of class II regulation. This transient and reversible experimental modulation of MHC class II expression in live cells by transcriptional intervention provides a new tool to study the function of class II molecules in various biological models.