The aquatic ecosystems of Doñana and those of its watersheds are highly threatened by multiple human pressures. Among them, the strong development of agriculture and the increase of population have been the main causes of the loss of quantity and quality of freshwaters during the last decades. Nitrate pollution is one of the main problems affecting both surface and groundwaters in Doñana and its surroundings. Previous studies have shown that the Doñana aquifer receives nitrates due to the infiltration of fertilizers in areas with intense agricultural activity. Furthermore, since 2008, part of the Doñana Natural Area (END) as well as other adjacent areas have been designated as "Nitrate Vulnerable Zones" according to the Nitrates Directive (91/676/EEC). However, historical data on surface water quality are scarce and existing data provide a low spatio-temporal resolution. In addition, very little information is available to date on the specific origin of nutrients reaching these aquatic systems. From a management point of view, the latter is essential when establishing appropriate management measures. In this context, the objective of this study focused on the characterisation of the nitrate pollutant sources and of the main transformation processes in surface waters of the watershed discharging into the Doñana marsh. concentration of nitrates (NO3-) were measured and the isotopic composition of nitrogen (?15N) and oxygen (?18O) were analysed in 29 samples of surface water collected between 2015 and 2016 from different streams and a lagoon located in three of the main sub-basins that drain into the Doñana marshes (La Rocina, El Partido and Los Sotos). Samples from Laguna Primera de Palos (Huelva), a pond located 35 km northwest of the National Park, were also included in order to obtain reference isotope values of an aquatic system affected almost exclusively by inorganic fertilizers, derived from strawberry and other red fruit greenhouse crops. Results showed that nitrate pollution in the study area comes mainly from agricultural fertilizers and treated wastewaters. The relative contribution of each of the contaminating sources was highly variable, depending on the sampling point and time of year, and is directly associated with the main land uses in the basin. Another important result derived from this study is that denitrification could be playing a key role in the natural removal of nitrates. According to the isotopic results obtained, it is estimated that between 50-75% of the nitrates that would potentially be received by the streams and lagoons studied, are previously denitrified. This means that water quality monitoring protocols that only measure nitrate concentrations in water would be underestimating the actual pollution caused by human activities in the basins. Given the strong intra- and inter-annual fluctuations in rainfall, which are characteristic of the Mediterranean climate, future studies would need to increase the spatio-temporal resolution of the data in order to obtain more precise information to help implement more adequately measures to reduce nitrate pollution in Doñana and its catchment areas. informacion[at]ebd.csic.es: Paredes et al (2020) Agricultural and urban delivered nitrate pollution input to Mediterranean temporary freshwaters. Agric. Ecosyst. Environ. 294, 106859. DOI 10.1016/j.agee.2020.106859