The p38 family of mitogen-activated protein kinases is believed to mediate a variety of leukocyte responses to pro-inflammatory stimuli. There are four members of the p38 family, and although activation of the different members has been studied in transiently transfected cells much less is known about activation of the endogenous p38s, particularly in myeloid lineage cells. To investigate activation of endogenous p38s, we have made monoclonal antibodies specific for each p38 and have used these antibodies to study p38 activation by pro-inflammatory stimuli in several human monocytic cell lines. Without stimulation endogenous p38alpha kinase activity was readily detectable, whereas that of p38beta, gamma, and delta was barely measurable. In response to inflammatory stimuli, we observed a time- and dose-dependent activation of all four p38s. The kinetics of activation of each of the p38s were similar for each stimulus used, suggesting a common upstream activation pathway. Simultaneous activation of the p38s suggests that all four may be important in inflammation.