Human monocyte chemotactic factor-3 (MCP-3) belongs to the C-C chemokines, which are cytokines involved in cell recruitment in inflammation and cancer. Northern blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that the MCP-3 gene is expressed in many human tissues and tumor cell lines and that the expression level is increased by various stimuli. Measles virus and phorbol 12-myristate 13-acetate (PMA) induced MCP-3 mRNA after 6 hr of stimulation. Interferon-beta (IFN-beta) induced MCP-3 mRNA after 16 hr, a time point when the PMA-induced mRNA had the tendency to level off. No significant increase in MCP-3 mRNA levels was observed in MG-63 cells after stimulation with interleukin-1beta (IL-1beta). To elucidate the regulation of MCP-3 gene expression, we determined the sequence of 5 kb of the MCP-3 promoter. This sequence contained a microsatellite that was shown to be polymorphic in various cell lines. Next 5'-deletion mutants of the promoter were generated and transfected into MG-63 cells, demonstrating the presence of several positive and negative transcriptional regulatory elements. One of the positive elements was located at -37, only 21 bp upstream from the TATAA box. This element was similar to an AP-1 element and also to a homeodomain protein Pbx1 binding site. A deletion mutant from -110 to +52 possessed the highest promoter activity, and the longer deletion mutants had relatively low activities. The region between -190 and -172 contained an Ets-like element and inhibited promoter activity. Stimulation with PMA dramatically increased promoter activity through activation of a positive element present between -172 and -100. The same 5'-deletion mutants were transfected into HeLa and Jurkat cells. None of the deletion mutants had any significant activity in Jurkat cells. In HeLa cells, low levels of MCP-3 mRNA were detected by RT-PCR, but the profile of the promoter activities of the deletion mutants was different from that seen in MG-63 cells.