Marine Crenarchaeota are among the most abundant groups of prokaryotes in the ocean and recent reports suggest that they oxidize ammonia as an energy source and inorganic carbon as carbon source, while other studies indicate that Crenarchaeota use organic carbon and hence, live heterotrophically. We used catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) to determine the crenarchaeal and bacterial contribution to total prokaryotic abundance in the (sub)tropical Atlantic. Bacteria contributed ∼50% to total prokaryotes throughout the water column. Marine Crenarchaeota Group I (MCGI) accounted for ∼5% of the prokaryotes in subsurface waters (100 m depth) and between 10 and 20% in the oxygen minimum layer (250–500 m depth) and deep waters (North East Atlantic Deep Water). The fraction of both MCGI and Bacteria fixing inorganic carbon, determined by combining microautoradiography with CARD-FISH (MICRO-CARD-FISH), decreased with depth, ranging from ∼30% in the oxygen minimum zone to < 10% in the intermediate waters (Mediterranean Sea Outflow Water, Antarctic Intermediate Water). In the deeper water masses, however, MCGI were not taking up inorganic carbon. Using quantitative MICRO-CARD-FISH to determine autotrophy activity on a single cell level revealed that MCGI are incorporating inorganic carbon (0.002–0.1 fmol C cell−1 day−1) at a significantly lower rate than Bacteria (0.01–0.6 fmol C cell−1 day−1). Hence, it appears that MCGI contribute substantially less to autotrophy than Bacteria. Taking the stoichiometry of nitrification together with our findings suggests that MCGI might not dominate the ammonia oxidation step in the mesopelagic waters of the ocean to that extent as the reported dominance of archaeal over bacterial amoA would suggest.