Abstract: Iron storage in yeast requires the activity of the vacuolar iron transporter Ccc1. Yeast with an intact CCC1 are resistant to iron toxicity but deletion of CCC1 renders yeast susceptible to iron toxicity. We used genetic and biochemical analysis to identify suppressors of high iron toxicity in ccc1 cells in order to probe the mechanism of high iron toxicity. All genes identified as suppressors of high iron toxicity in aerobically grown ccc1 cells encode organelle iron transporters including mitochondrial iron transporters MRS3, MRS4 and RIM2. Overexpression of MRS3 suppresses high iron toxicity by decreasing cytosolic iron through mitochondrial iron accumulation. Under anaerobic conditions ccc1 cells are still sensitive to high iron toxicity, but overexpression of MRS3 does not suppress iron toxicity and does not result in mitochondrial iron accumulation. We conclude that Mrs3/Mrs4 can sequester iron within mitochondria under aerobic conditions but not anaerobic conditions. We show that iron toxicity in ccc1 cells occurs under both aerobic and anaerobic conditions. Microarray analysis shows no evidence of oxidative damage under anaerobic conditions, suggesting that iron toxicity may not be solely due to oxidative damage. Deletion of TSA1 which encodes a periredoxin exacerbates iron toxicity in ccc1 cells under both aerobic and anaerobic conditions suggesting a unique role for Tsa1 in iron toxicity.