Methane seep deposits are common in the Upper Cretaceous Pierre Shale of the U.S. Western Interior. They contain a rich fauna including ammonites, bivalves, gastropods, sponges, corals, echinoids, crinoids, and fish. In an effort to understand the role of ammonites in these ecosystems, we examined a seep from the upper Campanian Didymoceras cheyennense Zone in Custer County, South Dakota, that contains molluscs with well-preserved shell material permitting isotopic analyses. Values of δ13C of the micritic limestone at the seep range from −46.94‰ to −11.49‰, confirming the influence of anaerobic oxidation of methane on the isotopic composition of the dissolved inorganic carbon reservoir. The ammonites also consistently display light values of δ13C ranging from −13.71‰ to 0.68‰. These values are generally lighter than those in nonseep specimens from age-equivalent rocks elsewhere in the basin (–1.75‰ to 3.42‰). In a single specimen of Baculites corrugatus from the seep, light δ13C values occur throughout ontogeny. These data suggest that ammonites incorporated isotopically light methane-derived carbon in their shells and lived near the vent fluids and methane-oxidizing bacteria. Both juvenile and adult specimens are present, implying that these ammonites spent their entire lives at the seep and formed an integral part of an interwoven community. The values of 87Sr/86Sr in the limestone and well-preserved fossils at the seep (0.707690–0.707728) are higher than that of the open ocean at this time (0.707659). These elevated values suggest that the seep fluids were imprinted with a radiogenic Sr signature, perhaps derived from equilibration with granitic deposits at depth during the initial uplift of the Black Hills.