The importance of spring thaw nitrous oxide (N2O) fluxes to the total N2O emission budget in cold climates has been recognized recently. Two mechanisms have been proposed to explain the burst in N2O fluxes due to soil freezing and thawing: enhanced microbial activity due to increased nutrient availability at spring thaw, and release of N2O trapped at depth during winter. The objective of this study was to determine whether increased surface N2O fluxes were due to physical release at spring thaw of N2O accumulated all winter at depth in the soil profile, or whether fluxes were due to rapid N2O production in the surface layer during the thaw process. Micrometeorological flux measurements and a chamber method applied to in situ soil columns receiving 15N tracer were used in Ontario, Canada during winters of 2003 and 2004. Labeled K15NO3 fertilizer (60% excess 15N) at the rate of 100 kg N ha−1 was applied to two layers, that is, surface layer 0 to 5 cm (SL) and deep layer 12 to 17 cm (DL) in nondisturbed soil columns placed in the field during the winter. The burst in N2O fluxes from the soil surface measured by both methods occurred within the same period of soil thawing. Denitrification was the main mechanism responsible for N2O production, and conditions conducive to N2O and N2 production occurred both in the SL and DL during thawing. Despite high 15N2O concentrations at depth, the burst in N2O fluxes from DL soil columns were 1.5 to 5 times lower than that from SL soil columns as more N2O from DL was converted to N2 before diffusing out of the soil profile. Comparison of N2O fluxes originating from SL and DL soil columns indicates that the source of N2O burst at spring thaw is mostly ‘newly’ produced N2O in the surface layer, and not the release of N2O trapped in the unfrozen soil beneath the frozen layers.