p38 mitogen-activated protein kinases (MAPKs) are activated primarily in response to inflammatory cytokines and cellular stress, and inhibitors which target the p38α and p38β MAPKs have shown potential for the treatment of inflammatory disease. Here we report the generation and initial characterization of a knockout of the p38β (MAPK11) gene. p38β^−/− mice were viable and exhibited no apparent health problems. The expression and activation of p38α, ERK1/2, and JNK in response to cellular stress was normal in embryonic fibroblasts from p38β^−/− mice, as was the activation of p38-activated kinases MAPKAP-K2 and MSK1. The transcription of p38-dependent immediate-early genes was also not affected by the knockout of p38β, suggesting that p38α is the predominant isoform involved in these processes. The p38β^−/− mice also showed normal T-cell development. Lipopolysaccharide-induced cytokine production was also normal in the p38β^−/− mice. As p38 is activated by tumor necrosis factor, the p38β^−/− mice were crossed onto a TNFΔARE mouse line. These mice overexpress tumor necrosis factor, which results in development symptoms similar to rheumatoid arthritis and inflammatory bowel disease. The progression of these diseases was not however moderated by knockout of p38β. Together these results suggest that p38α, and not p38β, is the major p38 isoform involved in the immune response and that it would not be necessary to retain activity against p38β during the development of p38 inhibitors.