Transcription of the nonmuscle myosin heavy chain II-A (NMHC-A) gene is regulated by various factors, including cell type, proliferation and differentiation stage, and extracellular stimuli. We have identified an intronic region (designated 32kb-150), which is located 32 kb downstream of the transcription start sites in the human NMHC-A gene, as a transcriptional regulatory region. 32kb-150 contains an interferon-stimulated response element (ISRE). By using HeLa and NIH3T3 cells, in which NMHC-A is constitutively expressed, interferon regulatory factor (IRF)-2 was found to be the only major protein, among the IRF family proteins, that bound to the ISRE in 32kb-150 both in vitro and in intact cells. IRF-2, which is known to either repress or activate target gene expression, acts as a transcriptional activator in the context of the 32kb-150 reporter gene. The carboxyl-terminal basic region of IRF-2 serves as an activation domain in this context. This is in contrast to its acting as a repressor domain in the context of the synthetic core ISRE. Furthermore, after treatment of promyelocytic HL-60 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), which triggers differentiation into macrophages, both NMHC-A expression and IRF-2 expression were found to be up-regulated with a similar time course. TPA treatment leads to recruitment of IRF-2 to 32kb-150 of the endogenous NMHC-A gene and acetylation of the core histones surrounding this region. In addition, the ISRE in the 32kb-150 reporter gene recruits IRF-2 and mediates TPA-induced activation of a reporter gene in HL-60 cells. Together, these results indicate that IRF-2 contributes to transcriptional activation of the NMHC-A gene via 32kb-150 during TPA-induced differentiation of HL-60 cells.