The transcriptional activation of the chicken lysozyme gene (cLys) by lipopolysaccharide (LPS) in macrophages is dependent on transcription of a LPS-Inducible Non-Coding RNA (LINoCR) triggering eviction of the CCCTC-binding factor (CTCF) from a negative regulatory element upstream of the lysozyme transcription start site. LINoCR is transcribed from a promoter originally characterized as a hormone response enhancer in the oviduct. Herein, we report the characterization of this cis-regulatory element (CRE). In activated macrophages, a 60 bp region bound by NF-kappaB, AP1 and C/EBPbeta controls this CRE, which is strictly dependent on NF-kappaB binding for its activity in luciferase assays. Moreover, the serine/threonine kinase IKKalpha, known to be recruited by NF-kappaB to NF-kappaB-dependent genes is found at the CRE and within the transcribing regions of both cLys and LINoCR. Such repartition suggests a simultaneous promoter and enhancer activity of this CRE, initiating cLys transcriptional activation and driving CTCF eviction. This recruitment was transient despite persistence of both cLys transcription and NF-kappaB binding to the CRE. Finally, comparing cLys with other LPS-inducible genes indicates that IKKalpha detection within transcribing regions can be correlated with the presence of the elongating form of RNA polymerase II or concentrated in the 3' end of the gene.