The West Florida Shelf, located in the eastern Gulf of Mexico, transitions from a eutrophic ecosystem dominated by the Mississippi River plume to mesotrophic and oligotrophic ecosystems off the coast of peninsular Florida. Three extensive trawl surveys in this region were used to acquire samples of fish muscle, benthic algae from sea urchin stomach contents, and filtered particulate organic matter (POM) to create delta C-13 and delta N-15 isoscapes. Muscle delta N-15 from three widely distributed fish species, Synodus foetens (inshore lizardfish), Calamus proridens (littlehead porgy), and Syacium papillosum (dusky flounder), exhibited strong longitudinal correlations (Pearson's r= -0.67 to -0.90, p <0.001) that coincided with the principal trophic gradient, whereas delta C-13 values of fish muscle and benthic algae were correlated with depth (Pearson's r=-0.34 to 0.73, p < 0.05). Correlations between isotopic values and surface concentrations of chlorophyll and particulate organic carbon (POC) imply linkages between the isotopic baseline and transitions from eutrophic to oligotrophic waters. The delta C-13 depth gradient and the delta N-15 longitudinal gradient were consistent between seasons and years, providing a foundation for future stable isotope studies of animal migration in the Gulf of Mexico. (C) 2013 Elsevier Ltd. All rights reserved.