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Anecdotal observations of the Dougherty plain cave crayfish ( Cambarus cryptodytes), the Georgia blind cave salamander ( Haideotriton wallacei), and albinistic isopods ( Caecidotea sp.) at great depths below the land surface and distant from river corridors suggest that obligate aquifer-dwelling (troglobitic) organisms are widely distributed throughout the limestone Upper Floridan aquifer (UFA). One mechanism by which subterranean life can proliferate in an environment void of plant productivity is through a microbial food web that includes chemosynthesis. We examined this possibility in the UFA by measuring the isotopic composition (13C, 14C, and 15N) of tissues from troglobitic macrofauna. Organisms that were captured by cave divers entering into spring conduits had δ13C values that suggested plant matter as a primary food resource (cave crayfish, –24.6 ± 2.7‰, n = 9). In contrast, δ13C values were significantly depleted in organisms retrieved from wells drilled into areas of the UFA remote from spring and sinkhole conduits (cave crayfish −34.7 ± 9.8‰, n = 10). Depleted 13C values in crayfish were correlated with radiocarbon (Δ14C) depletion relative to modern values. The results suggest that methane-based microbial chemosynthetic pathways support organisms living in the remote interior of the aquifer, at least in part. [ABSTRACT FROM AUTHOR]