NF-beta A is a monocyte, neutrophil, and B cell-specific nuclear protein that is involved in regulation of the IL-1 beta gene. These studies further define the functional role of NF-beta A in RAW264.7 monocytic cells by using transient transfection analysis. We showed that NF-beta A was able to activate transcription from a heterologous promoter in a distance-independent and dose-dependent manner. NF-beta A also appeared to function in a positionally independent manner within the IL-1 beta cap-site proximal (CSP) promoter. NF-beta A was required for maximal IL-1 beta gene expression directed by the upstream LPS-inducible enhancer element. Deletion of the NF-beta A-binding sequence resulted in an 80% reduction in basal reporter gene activity and an 86% reduction in LPS-inducible reporter gene activity in constructs containing only the enhancer and CSP promoter. Other regulatory elements located between the enhancer and the cap site were not able to substitute functionally for the absence of NF-beta A. Recently, other investigators have reported that IL-1 beta CSP promoter function was decreased by introducing multiple mutations within both the NF-beta A-binding sequence, and a putative overlapping NF-IL-6-binding sequence. We have found that these mutations predominantly affect NF-beta A binding. Furthermore NF-beta A, and not NF-IL-6, was required for supporting basal and LPS-inducible transcription from a minimal IL-1 beta CSP promoter (positions -58 to +11). This promoter region did not appear to direct monocyte-specific IL-1 beta gene expression because reporter constructs containing the IL-1 beta CSP promoter were also active in transiently transfected HeLa cells.