Microbial N 2 O release during the course of thawing of soil was investigated in model experiment focusing on denitrification, since freeze-thaw has been shown to cause significant physical and biological changes in soil, including a surge of N 2 O and CO 2 . The origin of these is still controversially discussed. The increase in denitrification after thawing may be attributed to the diffusion of organic substrates newly available to denitrifiers from disrupted soil aggregates, leading to an increase in microbial activity. Laboratory experiments with upper soil layer of a grassland were conducted in microcosms for real-time gas measurements during the entire phase of freeze and thaw. Shifts in microbial communities were evident on resolution of 16S and 18S rRNA genes and transcripts by denaturing gradient gel electrophoresis (DGGE). Microbial expression profiles were compared by RNA-arbitrarily primed PCR technique and subsequent resolution of amplified products on acrylamide gels. Differences in expression levels of periplasmic nitrate reductase gene (napA) and cytochrome cd 1 nitrite reductase (nirS) were observed by most-probable-number-reverse transcription-PCR, with higher levels of expression occurring just after thawing began, followed by a decrease. napA and nirS DGGE profiles showed no change in banding patterns with fingerprints derived from DNA, whereas those derived from cDNA showed a clear succession of denitrifying bacteria, with the most complex pattern being observed at the end of the N 2 O surge. This study provides insight into the structural community changes and expression dynamics of denitrifiers as a result of freeze-thaw stress. Also, the results presented here support the belief that the gas fluxes observed during thawing is a result of freezing initiated high microbial activity.The main process that forms the trace gas nitrous oxide (N 2 O) in agricultural soils is denitrification. It contributes significantly to the greenhouse effect. The concentration of N 2 O in the troposphere has increased from 270 ppb in 1750 to concentration of 316 ppb in the year 2000 and continues to rise (15). Moreover, it is involved in the destruction of stratospheric ozone. Therefore, identifying sources of N 2 O has been a subject of research over the last two decades. Several field studies in the temperate regions have indicated that N 2 O emissions in winter and spring, due to freezing and thawing of agricultural soils, can reach between 20 and 70% of the annual budget (28,35).