Thermophilic and hyperthermophilic microorganisms were cultured from 18°C diffuse hydrothermal fluids at the CoAxial segment deep-sea hydrothermal vent site 3 months after an eruption resulting from an intrusion of magma into shallow crust. The abundances of these organisms decreased over a 3-year period as the shallow magma cooled. The presence of these organisms at the site suggests that these organisms grew in response to nutrient input from hydrothermal fluid circulation and then were flushed to the surface following the eruption. Thermophiles and hyperthermophiles were also found in low-temperature (3–30°C) fluids at three other chronic, highly active deep-sea vent sites. The origin of these organisms is not known but may include the overlying seawater or a shallow to deep subseafloor habitat.