Grazing animals excrete urine and create transitorily high pH, nitrogen (N)-replete soil patches. Beneath grazed pasture, we postulated the soil microbial community would be highly responsive to N application. Lesser responses were expected of soils beneath grassland without grazing animals. Soil samples were collected near Lincoln, New Zealand (43.6°S, 172.5°E), beneath pasture regularly grazed by dairy cattle, an adjacent pasture set aside from grazing 20 years ago, and a nearby grassland that has never been grazed. Soil microbial respiration responses to repeated urea (500 kg N/ha) applications were determined by laboratory incubation experiments and the soil pH and water-soluble C content were also measured. The first application induced 0.13 ± 0.04 (dairy farm), 0.15 ± 0.05 (set aside), and 0.20 ± 0.04 (ungrazed) g C/kg increases in microbial respiration over 9 days, excluding carbon dioxide production from carbonate hydrolysis. After a second application, 9-day respiration increased by 0.26 ± 0.04 (dairy farm), 0.41 ± 0.04 (set aside) and 0.20 ± 0.07 (ungrazed) g C/kg. For the dairy farm and ungrazed soils, the microbial communities responded differently to repeated urea addition. The responses included transitory changes in pH and reflected the limited amounts of readily decomposable organic matter.