Interleukin 1β (IL-1β) is a proinflammatory cytokine that exhibits a wide variety of biological activities. Genomic sequences that mediate the induction of human IL-1β gene transcription by lipopolysaccharide and phorbol esters are located more than 2,700 bp upstream of the transcriptional start site (cap site). These upstream elements require additional cap site-proximal (CSP) sequences which are necessary for basal transcription of the human IL-1β gene. In addition, these CSP sequences have been shown to mediate both cell type-specific expression of this gene, and trans-activation by some viral proteins. In this study, we report the identification of a novel nuclear protein, termed NFβC, that binds to a DNA sequence which spans the cap site of the human IL-1β gene (positions −12 to +8). We have also identified a second region (positions −305 to −280) containing a putative NF-κB binding site. We show here that this region can bind three distinct nuclear proteins. One protein is similar or identical to NF-κB, a second protein (termed NFβB) binds a distinct sequence that substantially overlaps the 5' half of the NFκB binding sequence, and a third protein (termed NFβD) binds a distinct sequence that substantially overlaps the 3' half of the NFκB binding sequence. Unlike NFκB, NF1βB and NFβD are present in nuclear extracts prepared from unstimulated monocytic cells. Although the NFβD and NFβC binding sequences share no significant similarity, each sequence can specifically compete for the binding of either protein to DNA, whereas oligonucleotides containing only the NFκB or NFβB motifs do not compete for the binding of NFβC or NFβD. This suggests that NFβC and NFβD can specifically interact in vitro, possibly through a common subunit.