Abstract: The antiarrhythmic drug amiodarone has fungicidal activity against a broad range of fungi. In S. cerevisiae, it elicits an immediate influx of Ca2+ followed by mitochondrial fragmentation and eventual cell death. To dissect the mechanism of its toxicity, we assessed the transcriptional response of S. cerevisiae to amiodarone by DNA microarray. Consistent with the drug induced calcium burst, more than half of the differentially transcribed genes were induced by high levels of CaCl2. Amiodarone also caused rapid nuclear accumulation of the calcineurin regulated Crz1. The majority of genes induced by amiodarone within 10 minutes were involved in utilization of alternative carbon and nitrogen sources and in mobilizing energy reserves. The similarity to nutrient starvation responses seen in stationary phase cells, rapamycin treatment and late stages of shift to diauxic conditions and nitrogen depletion suggests that amiodarone may interfere with nutrient sensing and regulatory networks. Transcription of a set of nutrient-responsive genes was affected by amiodarone but not CaCl2, indicating that activation of the starvation response was independent of Ca2+. Genes down-regulated by amiodarone were involved in all stages of cell cycle control. A moderate dose of amiodarone temporarily delayed cell cycle progression at G1, S and G2/M phases, with the Swe1-mediated delay in G2/M phase being most prominent in a calcineurin-dependent manner. Overall, the transcriptional responses to amiodarone revealed by this study were found to be distinct from other classes of antifungals, including the azole drugs, pointing towards a novel target pathway in combating fungal pathogenesis.