Spleen cells from male adult BALB/c mice given intraperitoneal injections of purified coxsackievirus B-3 were examined for the ability to lyse syngeneic neonatal myofibers in culture. Cytotoxicity against infected and uninfected targets was measured with the use of an in vitro 51Cr release assay. Immune spleen cells obtained 4--7 days after infection were cytotoxic for viral-infected myofibers. Peak reactivity was observed 5 days after infection. At this time immune spleen cells showed significantly less reactivity against uninfected myofibers. Cytotoxicity against infected targets was mediated by T lymphocytes, since reactivity was abolished by treatment with anti-thy 1.2 and complement. Treatment with anti-Ig and complement caused no loss of activity. Reciprocal assays performed with BALB/c and CBA cells showed that maximal cytotoxicity occurred against infected syngeneic myofibers, providing further evidence that viral-specific effector cells were T lymphocytes. In addition, hyperimmune rabbit anti-coxsackievirus B-3 antiserum could not block immune spleen cell lysis of infected targets, suggesting that coxsackievirus-infected myofibers expressed surface membrane antigens not recognized by specific neutralizing antibody.