Background and Aims: Roots in agroecosystems encounter spatially and temporally heterogeneous nitrogen (N) availability in soil. Understanding root physiological processes in concert with soil microbial N dynamics following spatially discrete N pulses under field conditions will aid in the management of agroecosystem processes for N use efficiency. Methods: This study examined the short-term response (<5 days) of tomato (Solanum lycopersicum L.) roots and soil N cycling to a pulse of inorganic N in an undisturbed soil patch on an organic farm using a novel combination of molecular and 15N isotopic techniques. Results: Tomato roots rapidly responded to and exploited the N pulse via upregulation of key N metabolism genes (e.g. cytosolic glutamine synthetase GS1) that comprise the core physiological response of roots to patchy soil N availability. Strong root activity limited accumulation of soil NO3- despite high rates of gross nitrification. Roots out-competed soil microbes for the inorganic N, even on a short time scale, likely as a result of high plant N demand and microbial C limitation. The transient root gene expression response (absent by 4 days after the N pulse) underscored the sensitivity of root N uptake to local N availability. Conclusions: Root expression of genes such as GS1 could complement soil inorganic N pools and measurements of soil microbial activity to serve as integrative indicators of rapid plant-soil N cycling.