In activated macrophages, the rel/NF-kappaB transcription factors are known to play important roles in interleukin-12 (IL-12) p40 regulation by nitric oxide (NO). However, the relative contributions of these factors are not well understood. Here, we describe a dominant role for c-rel involving p38 mitogen-activated protein kinase (p38 MAPK) and calmodulin (CaM) protein in NO-mediated IL-12 p40 inhibition in activated macrophages. Inhibition of NO production by aminoguanidine increased, whereas sodium nitroprusside (SNP; an exogenous NO generator) reduced, nuclear c-rel levels in LPS + IFN-gamma-activated RAW 264.7 macrophages. Overexpression of c-rel but not p65 NF-kappaB increased IL-12 p40 during NO treatment. The p38 MAPK phosphorylation is increased by NO, and inhibition of p38 MAPK in SNP-treated macrophages by SB203580 or transient expression of a dominant-negative mutant of p38 MAPK upregulated both nuclear c-rel and IL-12 p40 levels, indicating that NO targeted the p38 MAPK pathway to inhibit c-rel and IL-12 p40. Cytoplasmic CaM level was increased by NO, and SB203580 decreased the CaM level in NO-exposed macrophages. Inhibition of CaM activity by trifluoperazine rescued the inhibitory effect of NO on c-rel and IL-12 p40. Our findings indicate that c-rel plays an important role in NO-mediated inhibition of IL-12 p40 and is regulated by p38 MAPK through CaM protein.