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Changes in soil moisture with cycles of soil wetting and drying are associated with shifts in osmotic potentials that can induce physiological stress for microbial communities. These instances of soil moisture stress can be of sufficient magnitude to alter flows of C and N at an ecosystem scale. In this study we manipulated the duration and severity of soil moisture stress and disturbance in grassland soils from four sites along a precipitation gradient. After subjecting soils to a two-month long incubation under two different wetting-drying regimes, one of high and one of low stress and disturbance, we moistened soils with C-13- and N-15-labeled glycine solution to trace C and N though the soil and its microbial communities as they dried. Contrary to our predictions, we found evidence for preferential use of N-free osmolytes with increased soil moisture stress in soils from the mesic end of the precipitation gradient. Soils from the western, semi-arid end of the gradient were less sensitive to soil moisture stress and did not differ in N demand under high and low stress. Specific respiration rates were higher in all soils under greater soil moisture stress immediately after re-wetting, then returned to levels equal to or below rates in soils under low soil moisture stress regimes. Nitrification outpaced denitrification processes in soils under the highest levels of soil moisture stress. These results suggest increases in both soil CO2 release and N losses as stress induced by greater soil moisture variability increases in relatively mesic grassland systems, a predicted consequence of climate change in this region. (c) 2012 Elsevier Ltd. All rights reserved.