The temporal and spatial distribution of active c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) in the brain was investigated in an experimental virus-mouse system in which neurovirulent influenza A virus caused lethal acute encephalitis. Following stereotaxic microinjection into the olfactory bulb, virus-infected neurons appeared in several midbrain structures, including the ventral tegmental area, amygdala and the pyramidal layer of the hippocampus. Infected neurons exhibited apoptosis on day 5, as demonstrated by in situ detection of DNA fragmentation and active caspase-3. The stress-responsive JNK signal transduction pathway was activated in virus-infected neurons. Activation of p38 MAPK was widespread and occurred in astrocytes on day 7 after infection. Active p38 MAPK in astrocytes showed no association with apoptosis but appeared to be involved in regulation of TNF-alpha production. These results indicate that these two stress-activated protein kinases may play distinct roles during the course of lethal acute influenza virus encephalitis.