Interleukin-1beta (IL-1beta) induces the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. However, the molecular regulation of IL-1beta expression in myeloid differentiation has not been elucidated. In this study the chromatin structure of the IL-1beta promoter and the impact of methylation on IL-1beta expression in monocytic development were examined. The results revealed that the IL-1beta promoter was inaccessible in undifferentiated promyeloid HL-60 cells but highly accessible in differentiated monocytic cells which additionally acquired the ability to produce IL-1beta. Accessibilities of differentiated cells were comparable to those of primary monocytes. Lipopolysaccharide (LPS) stimulation did not affect promoter accessibility in promyeloid and monocytic HL-60 cells, demonstrating that the chromatin remodelling of the IL-1beta promoter depends on differentiation and not on the transcriptional status of the cell. Demethylation via 5-aza-2'-deoxycytodine led to the induction of IL-1beta expression in undifferentiated and differentiated cells, which could be increased after LPS stimulation. Our data indicate that the IL-1beta promoter is reorganized into an open poised conformation during monopoiesis being a privilege of mature monocytes but not of the entire myeloid lineage. As a second mechanism, IL-1beta expression is regulated by methylation acting independently of the developmental stage of myeloid cells.