Interleukin-8 (IL-8), a potent neutrophil chemoattractant, can be expressed at high levels by many different cell types after immune stimulation. In contrast, expression of IL-8 in these same cells is virtually absent in the unstimulated state, demonstrating the tight regulation of the IL-8 gene. Although much is known about how this gene is transcriptionally activated after immune stimulation, little is known about the regulation of the IL-8 promoter in the absence of immune activation. In this study we examine how the IL-8 promoter is transcriptionally regulated in the uninduced state and how these mechanisms are altered in response to immune stimulation by IL-1beta. Electrophoretic mobility shift assay and transfection studies show that the IL-8 promoter is transcriptionally regulated by both positive and negative elements. Although the nuclear factor-kappaB (NFkappaB) element regulates only inducible activity of the IL-8 promoter in response to stimulation with IL-1beta, the AP-1 and CCAAT/Enhancer-binding Protein (C/EBP) elements influence both basal and inducible activities. In contrast to these three positive regulatory elements, the binding of the ubiquitously expressed POU-homeodomain transcription factor, Oct-1, strongly represses transcriptional activity of the IL-8 promoter by binding independently to an element overlapping that of C/EBP.