Despite 40 years of research, knowledge of in situ metabolism of microbes carrying out carbon fixation in marine hydrothermal systems is still very limited. Particularly lacking are studies identifying the chemosynthetically active microbes and measuring rates of CO2 fixation in situ. Here, we present data from innovative incubation approaches for assessing chemoautotrophic production in deep-sea vent systems. We quantified chemosynthetic primary productivity of natural microbial communities sampled from Crab Spa (9oN East Pacific Rise) during incubations that maintained seafloor pressure and temperature.