IL-16, a leukocyte chemoattractant factor (LCF), is involved in the disease process of multiple sclerosis and other autoimmune disorders. However, mechanisms by which this LCF is expressed are poorly understood. The present study underlines the importance of IL-12 p40 homodimer (p40(2)), the so-called biologically inactive molecule, in inducing the expression of IL-16 in primary mouse and human microglia, mouse BV-2 microglial cells, mouse peritoneal macrophages, and RAW264.7 cells. In contrast, IL-12 p70, the bioactive heterodimeric cytokine, was unable to induce the expression of IL-16 in any of these cell types. Similarly IL-12 p40(2) also induced the activation of IL-16 promoter in microglia. Among various stimuli tested, p40(2) was the most potent one followed by p40 monomer, IL-16 and IL-23 in inducing the activation of IL-16 promoter in microglial cells. Furthermore, induction of IL-16 mRNA expression by over-expression of p40, but not p35, cDNA and induction of IL-16 expression by p40(2) in microglia isolated from IL-12p35 (-/-) mice confirm that p40, but not p35, is responsible for the induction of IL-16. Finally, by using primary microglia isolated from IL-12Rbeta1 (-/-) and IL-12Rbeta2 (-/-) mice, we demonstrate that p40(2) induces the expression of this LCF via IL-12Rbeta1 but not IL-12Rbeta2. These results delineate a novel biological function of p40(2) and raise the possibility that biological function of IL-12 p40(2) may be different from IL-12 p70.