The Adirondack Mountain region is an extensive geographic area (26,305 km2) in upstate New York where acid deposition has negatively affected water resources for decades and caused the extirpation of local fish populations. The water quality decline and loss of an established brook trout (Salvelinus fontinalis [Mitchill]) population in Brooktrout Lake were reconstructed from historical information dating back to the late 1880s. Water quality and biotic recovery were documented in Brooktrout Lake in response to reductions of S deposition during the 1980s, 1990s, and 2000s and provided a unique scientific opportunity to re-introduce fish in 2005 and examine their critical role in the recovery of food webs affected by acid deposition. Using C and N isotope analysis of fish collagen and state hatchery feed as well as Bayesian assignment tests of microsatellite genotypes, we document in situ brook trout reproduction, which is the initial phase in the restoration of a preacidification food web structure in Brooktrout Lake. Combined with sulfur dioxide emissions reductions promulgated by the 1990 Clean Air Act Amendments, our results suggest that other acid-affected Adirondack waters could benefit from careful fish re-introduction protocols to initiate the ecosystem reconstruction of important components of food web dimensionality and functionality.