Macrophage inflammatory protein-1α (MIP-1α) is produced in high concentration by multiple myeloma (MM) cells in about 70% of patients, and MIP-1α levels correlate with their disease activity. Patients who have high levels of MIP-1α have a poor prognosis. Furthermore, blocking MIP-1α expression in an in vivo model of human MM profoundly decreases both tumor burden and bone destruction, suggesting that MIP-1α is an important mediator of MM bone disease. Therefore, to analyze the regulation of MIP-1α production in MM, we cloned the human MIP-1α promoter and characterized the transcription factor (TF) motifs that control MIP-1α expression in MM cells. The proximal region of MIP-1α promoter was composed of 2 sets of identical transcription regulatory regions consisting of GATA-2⁺ AML-1⁺ C/EBPα motifs. Since 2 alternatively spliced variants of the acute myeloid leukemia-1 (AML-1) class of TFs can bind the AML-1 region, AML-1A and AML-1B, the relationship between the expression levels of AML-1A or AML-1B in MM cells and their capacity to express MIP-1α was examined. AML-1A mRNA was relatively overexpressed compared with AML-1B in MM cell lines that produced high levels of MIP-1α (> 1 ng/mL per 10⁶ cells per 72 hours), but AML-1A was not increased in MM cell lines that expressed less than 200 pg/mL MIP-1α. More importantly, the ratio of AML-1A to AML-1B mRNA levels was also increased in 3 of 3 highly purified myeloma cells from patients with MM who expressed increased amounts of MIP-1α. The ratio of AML-1A to AML-1B mRNA in patients with MM was 8-fold higher than in healthy controls. Transduction of AML-1B into the MM-derived MM.1S and ARH-77 cells totally blocked MIP-1α production, while AML-1A did not further increase the already high levels of MIP-1α produced by these cells. Taken together, these data demonstrate that in patients with MM who produce increased concentrations of MIP-1α, the relative level of AML-1B is significantly decreased compared with healthy controls. The data suggest that strategies that enhance AML-1B expression or decrease AML-1A in MM cells may be beneficial therapeutically.