Stable isotopes can be used to elucidate ecological relationships in community and trophic studies. Findings are calibrated against baselines, e.g. from a producer or primary consumer, assumed to act as a reference to the isotopic context created by spatio-temporal attributes such as geography, climate, nutrient, and energy sources. The ability of an organism to accurately represent a community base depends on how, and over what time-scale, it assimilates ambient materials. Freshwater mussels have served as references for trophic studies of freshwater communities and as indicators of change in nutrient pollution load or source. Their suitability as reference animals has not yet been fully explored, however. We conducted a series of studies examining the suitability of freshwater mussels as isotopic baselines, using their ability to reflect variation in ambient nutrient loads as a case scenario. (1) We analyzed bivalve foot tissue delta(15)N and delta(13)C from 22 stream reaches in the Piedmont region of North Carolina, USA to show that compositions varied substantially among locations. Site mean bivalve delta(13)C values correlated with site ambient particulate organic matter (POM) delta(13)C values, and site mean bivalve delta(15)N values correlated with site ambient water dissolved delta(15)N-NO(3) values. (2) Similarity of results among sample types demonstrated that the minimally invasive hemolymph sample is a suitable substitute for foot tissue in delta(15)N analyses, and that small sample sizes generate means representative of a larger population. Both findings can help minimize the impact of sampling on imperiled freshwater mussel populations. (3) In a bivalve transplantation study we showed that hemolymph delta(15)N compositions responded to a shift in ambient dissolved delta(15)N-NO(3), although slowly. The tissue turnover time for bivalve hemolymph was 113 days. We conclude that bivalves serve best as biomonitors of chronic, rather than acute, fluctuations in stream nutrient loads, and provide initial evidence of their suitability as time-integrated isotopic baselines for community studies.