The responses of primary cultures of rat myocardial cells to diazepam (Valium) were evaluated. The cells were exposed to 4, 16 or 32 microgram/ml of diazepam for 1, 4 or 24 h. The cultures were evaluated for changes in beating activity and morphology and for cytotoxic effects. After the 1-h treatment exposure, the cultures exhibited tachycardia which was dose-dependent. The longer durations of treatment either produced arrhythmias or caused a loss of beating. Major morphological alterations observed were the formation pseudopodia and increased cytoplasmic granulation. Cytotoxicity was measured quantitatively by leakage of lactic dehydrogenase (LDH) from the cells into the media and by cell viability. Moderate amounts of LDH were leaked from the cells after exposure to diazepam for 4 h. After 24 h of treatment with the higher concentrations of diazepam, cell viability was greatly reduced.