Eastern equine encephalitis virus (EEEV) is an arbovirus endemic to the eastern United States. Human cases are rare but can be serious. The primary enzootic vector is Culiseta melanura (Coquillett) (Diptera: Culicidae), an ornithophagic mosquito. We conducted an aerial application of a granular methoprene formulation in Hockomock Swamp (Massachusetts), which represents a focus of EEEV transmission. Water collected from inside and outside Cs. melanura crypts was evaluated in bioassays of early fourth instar Cs. melanura larvae using treated and untreated water. Adult eclosion rates were 36% significantly lower in treated compared with untreated water (P < 0.05). Eclosion rates for water collected from inside crypts were significantly higher (62%) than rates from outside crypts (30%) (P < 0.05), indicating higher efficacy outside crypts. We tested whether reduced methoprene efficacy inside the crypts was due to reduced chemical penetration into this habitat. Chemical water analyses confirmed that methoprene concentrations were lower inside the crypts (0.1 ± 0.05 ppb) compared to water from outside crypts (1.79 ± 0.41 ppb). The susceptibility of Cs. melanura to methoprene was also determined to allow for comparison against concentrations observed in water collected from the field (LC-95: 1.95 ± 0.5 ppb). Overall, methoprene-treated water prevented mosquito development for up to 4 wk, but with a reduction in efficacy between 4- and 6-wk post-application. Our results suggest that aerial methoprene applications can effectively treat open water in wetlands but may not provide efficacious control of Cs. melanura due to an inability to penetrate larval habitats.