The chemokine RANTES/CCL5 is a proinflammatory agent produced by a variety of tissues in response to specific stimuli. In human monocytes, RANTES/CCL5 transcription is up-regulated rapidly and transiently in response to LPS. We describe here two regions that help control LPS-driven transcription from the human RANTES/CCL5 promoter in monocytic cells. These sites were analyzed by using DNase I footprinting, transient transfection assays, site-directed mutagenesis, and EMSA. RANTES site E (R(E), -125/-99) constitutively binds C/EBP proteins in monocytic Mono Mac 6 cells. Mutation of region R(E) led to a significant (40%-50%) reduction in LPS-induced promoter reporter activity. Region R(AB) is composed of tandem kB-like elements R(A) and R(B) (-73/-34). These sites working in concert act as an LPS-responsive promoter module. R(A) constitutively binds Sp1, and Rel p50/p65 following LPS stimulation. Either factor can mediate transcriptional effects at R(A). Induced Rel p50/p50 binding to site R(B) is required for LPS regulation of RANTES/CCL5 transcription. A series of computer models based on the RANTES/CCL5 promoter were generated to represent the organization of these functional elements. The models could identify LPS-regulated promoters in human, other vertebrate, and viral sequences in various databases.