Nitrogen (N) is considered as a key element that triggers algal boom in the Indian River Lagoon (IRL), South Florida. Intensive agriculture may have contributed to increased N input into the IRL. Runoff and storm water samples were collected in representative agricultural fields and along waterways that connect lands to the IRL from April 2013 to December 2014. Concentrations of different N species (particulate N, dissolved organic N, dissolved NH4 (+)-N, and NO3 (-)-N) and related water physical-chemical properties were measured. Total N (TN) concentrations generally decreased from agricultural field furrows to discharging point of the waterways but were generally above the US EPA critical level (0.59 mg L(-1)) for surface water. Organic N was the dominant form of dissolved N, followed by NO3 (-)-N, and dissolved NH4 (+)-N. Concentrations and speciation of N in water varied with sites and sampling times but were generally higher in summer and fall and lower in spring and winter, as affected by the seasonality of regional hydrology and agricultural practices. Correlations occurred between N concentration, water physical properties, and rainfall. This information has important implications in the development of best management practices to minimize the impacts of agricultural practice on N loading in the Indian River Lagoon.