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It was suggested that the abnormally high N2O values found in 130,000-160,000 year-old Vostok ice core samples, characterized by high delta 15N and low delta 18O values, resulted from in situ microbial N2O production. To substantiate these observations we obtained new geochemical data from the last glacial period and showed the existence of additional small N2O anomalies. To test the hypothesis that microbial metabolism could contribute to these anomalies, we developed protocols for examining the ability of Nitrosomonas cryotolerans cells to produce N2O at subfreezing temperatures. Our results show that these model, frozen cultures produce N2O at temperatures as low as -32°C.