Caspase activation and MAP kinase signaling have been implicated in coxsackievirus B3 (CVB3) pathogenesis, and both have been demonstrated late in the virus life cycle. We studied activation of caspase-3, an effector protease of apoptosis, and ERK phosphorylation, indicative of MAPK signaling pathway activation, following CVB3 infection of cells that express the coxsackievirus and adenovirus receptor (CAR) or CAR constructs lacking the cytoplasmic domain, and cells which express no detectable CAR. These experiments showed that a burst of caspase-3 activity preceded lysis of CVB3-infected cells expressing CAR, irrespective of the CAR cytoplasmic domain. In RD cells, which were infected in the absence of detectable CAR, caspase-3 activity increased progressively over 52 h with no apparent burst. ERK phosphorylation also occurred late in the virus life cycle, preceding caspase-3 activation, and occurred in cells expressing full-length CAR but not in RD. These results show that ERK phosphorylation precedes caspase-3 activation, both occur late in the infection, and both are influenced by the presence of CAR.