OBJECTIVE: The evaluation of S100B protein expression in the human heart and its correlation with drug-related death. METHOD: Left ventricular samples were collected from 74 serial forensic autopsies (15 overdose-related deaths; 59 non-overdose-related deaths) from 2007 to 2010. Tissue sections from each sample were immunostained for S100B protein by a commercial antibody. RESULTS: The S100B protein was detected in the heart samples of all 15 cases of drug-related deaths; S100B immunoreactivity was mainly observed in the cytoplasm of cardiomyocytes and as globular deposits in the interstitial spaces. No reactivity or weak reactivity was found in the cardiomyocytes of the 59 subjects who died of other causes. CONCLUSION: Our preliminary data show that the S100B protein accumulates in injured cardiomyocytes during drug-related sudden death. Given the near absence of S100B protein in the heart of subjects who died from causes other than drug overdose, S100B immunopositivity may be used as a new ancillary screening tool for the postmortem diagnosis of overdose-related cardiac death.