Many karst regions are undergoing rapid population growth and expansion of urban land accompanied by increases in wastewater generation and changing patterns of nitrate (NO3(-)) loading to surface and groundwater. We investigate variability and sources of NO3(-) in a regional karst aquifer system, the Edwards aquifer of central Texas. Samples from streams recharging the aquifer, groundwater wells, and springs were collected during 2008-12 from the Barton Springs and San Antonio segments of the Edwards aquifer and analyzed for nitrogen (N) species concentrations and NO3(-) stable isotopes (δ(15)N and δ(18)O). These data were augmented by historical data collected from 1937 to 2007. NO3(-) concentrations and discharge data indicate that short-term variability (days to months) in groundwater NO3(-) concentrations in the Barton Springs segment is controlled by occurrence of individual storms and multi-annual wet-dry cycles, whereas the lack of short-term variability in groundwater in the San Antonio segment indicates the dominance of transport along regional flow paths. In both segments, longer-term increases (years to decades) in NO3(-) concentrations cannot be attributed to hydrologic conditions; rather, isotopic ratios and land-use change indicate that septic systems and land application of treated wastewater might be the source of increased loading of NO3(-). These results highlight the vulnerability of karst aquifers to NO3(-) contamination from urban wastewater. An analysis of N-species loading in recharge and discharge for the Barton Springs segment during 2008-10 indicates an overall mass balance in total N, but recharge contains higher concentrations of organic N and lower concentrations of NO3(-) than does discharge, consistent with nitrification of organic N within the aquifer and consumption of dissolved oxygen. This study demonstrates that subaqueous nitrification of organic N in the aquifer, as opposed to in soils, might be a previously unrecognized source of NO3(-) to karst groundwater or other oxic groundwater systems.