It is generally accepted that DNA conserves its secondary structure after a freeze-thaw cycle. A negligible amount of degradation occurs after this procedure. Degradation becomes appreciable only after multiple cycles of freezing and thawing. In this study, we have found that a single freeze-thaw cycle in alkaline medium (pH ≥ 10.8) gives rise to denaturation of calf thymus DNA, although the melting temperature of intact DNA in the solution used for the freeze-thaw experiments is higher than 60 °C. The degree of denaturation is almost independent of the regime of freezing. The melting curve obtained after DNA is frozen at -2 °C and then thawed is almost the same as after a freezing carried out in liquid nitrogen (-196 °C). However, incubation in the same solution at 0 °C for 24 hours without freezing does not give rise to any denaturation. The degree of denaturation caused by freezing increases with pH (if pH ≥ 10.8) and decreases with Na₂CO₃ concentration at fixed pH and [Na⁺], although Na₂CO₃ decreases the melting temperature of intact DNA. A preliminary treatment of DNA with cisplatin or transplatin (0.01 Pt atoms per nucleotide) gives rise to a full recovery of the DNA secondary structure after freezing and thawing similar to what occurs after heating DNA to 100 °C and quick cooling. Possible mechanisms that may cause DNA denaturation during a freeze-thaw cycle in alkaline medium are discussed.