The p38 mitogen‐activated protein kinase (MAPK) pathway, like the c‐Jun N‐terminal kinase (JNK) MAPK pathway, is activated in response to cellular stress and inflammation and is involved in many fundamental biological processes. To study the role of the p38 MAPK pathway in vivo, we have used homologous recombination in mice to inactivate the Mkk3 gene, one of the two specific MAPK kinases (MAPKKs) that activate p38 MAPK. Mkk3−/− mice were viable and fertile; however, they were defective in interleukin‐12 (IL‐12) production by macrophages and dendritic cells. Interferon‐γ production following immunization with protein antigens and in vitro differentiation of naive T cells is greatly reduced, suggesting an impaired type I cytokine immune response. The effect of the p38 MAPK pathway on IL‐12 expression is at least partly transcriptional, since inhibition of this pathway blocks IL‐12 p40 promoter activity in macrophage cell lines and IL‐12 p40 mRNA is reduced in MKK3‐deficient mice. We conclude that the p38 MAP kinase, activated through MKK3, is required for the production of inflammatory cytokines by both antigen‐presenting cells and CD4+ T cells.