Expression of intracellular ammonium assimilation enzymes were used to assess the response of nitrogen (N) metabolism in bacterioplankton to N-loading of sub-tropical coastal waters of Key West, Florida. Specific activities of glutamine synthetase (GS) and total glutamate dehydrogenase (GDHT) were measured on the bacterial size fraction (<0.8 microm) to assess N-deplete versus N-replete metabolic states, respectively. Enzyme results were compared to concentrations of dissolved organic matter and nutrients and to the biomass and production of phytoplankton and bacteria. Concentrations of dissolved inorganic N (DIN), dissolved organic N (DON), and dissolved organic carbon (DOC) positively correlated with specific activities of GDHT and negatively correlated with that of GS. Total dissolved N (TDN) concentration explained 81% of variance in bacterioplankton GDHT:GS activity ratio. The GDHT:GS ratio, TDN, DOC, and bacterial parameters decreased in magnitude along a tidally dynamic trophic gradient from north of Key West to south at the reef tract, which is consistent with the combined effects of localized coastal eutrophication and tidal exchange of seawater from the Southwest Florida Shelf and Florida Strait. The N-replete bacterioplankton north of Key West can regenerate ammonium which sustains primary production transported south to the reef. The range in GDHT:GS ratios was 5-30 times greater than that for commonly used indicators of planktonic eutrophication, which emphasizes the sensitivity of bacterioplankton N-metabolism to changes in N-bioavailability caused by nutrient pollution in sub-tropical coastal waters and utility of GDHT:GS ratio as an bioindicator of N-replete conditions.