Urine patches in grazed systems are hotspots for nitrogen (N) cycling and losses to the wider environment. Retention and subsequent recycling of urinary-N is key to minimise losses and increase ecosystem nitrogen use efficiency. Biosynthesis into the microbial organic N pool is an important N pathway but this has not been directly quantified in a urine patch. Herein, we present the results of a time course experiment using soil mesocosms sown with perennial ryegrass (Lolium perenne L.) and treated with 15N-labelled sheep urine to determine partitioning of the applied N between plant, soil biomass pools and leaching losses following simulated rainfall events. 15N-tracing used bulk and compound-specific 15N-stable isotope probing (SIP) to determine the fate of urinary N. Initial high leaching losses (233 kg N ha−1) were comprised of native soil N, ammonium and nitrate derived from urine by urea hydrolysis and nitrification, respectively. Leaching subsequently decreased whilst uptake into plant biomass and microbial biosynthesis increased during periods of low rainfall. Uptake into above and belowground plant biomass was the largest fate of urinary-15N after 94 d (42%), although assimilation into microbial biomass dominated for ca. 1 month after urine deposition (34%). Compound-specific 15N–SIP of amino acids and amino sugars revealed immobilisation of urinary-N following mineralisation was the dominant pathway for biosynthesis, with incorporation into bacterial organic N pools more rapid than into the fungal biomass. There was also intact utilisation of glycine derived from urine. This study provides clear evidence that direct assimilation of urine-derived N into microbial organic N pools is an important process for retaining N in a urine patch, which will subsequently support plant N supply during microbial turnover.