Characterizing and quantifying metabolism in situ remains both a central goal and challenge for environmental microbiology. Here,we used a single-cell, multi-isotope approach to investigate the anabolic activity of microorganisms in the Pacific Ocean, andspecifically the carbon assimilatory capabilities of natural populations of Thaumarchaeota. We incubated seawater from coastaleuphotic and aphotic zones with 13C-labeled inorganic and 15N-labeled organic substrates, and detected microbial uptake usingnanoscale secondary ion mass spectrometry (nanoSIMS). We performed high-throughput metabolic screening of 1,501 individual cellsand determined the percentage of anabolically active cells, the distribution of cellular activity across the community, and themetabolic lifestyle—chemoautotrophic or heterotrophic—of each cell. Most cells (>90%) demonstrated anabolic activity during theincubation, indicating that dormant cells are rare in marine water. While heterotrophy dominated both samples,chemoautotrophy was also detected, with 4-17% of cells acquiring carbon primarily from bicarbonate. When bacteria wereinhibited by antibiotics, diverse Marine Group I Thaumarchaeota increased in relative abundance (16S RNA genes) and activity (16SrRNA), as did the fraction of chemoautotrophic cells detected via nanoSIMS, suggesting chemoautotrophy by Thaumarchaeota. Weused fluorescence in situ hybridization coupled to nanoSIMS (FISH-nanoSIMS) to directly investigate Thaumarchaeota this confirmedwidespread chemoautotrophy by Thaumarchaeota, and a lack of chemoautotrophy in sampled bacteria. FISH-nanoSIMS of cellsincubated with dual-labeled (13C,15N-) amino acids showed enrichment of both 13C and 15N in bacteria, but enrichment of only 15Nin Thaumarchaeota. Contrary to previous studies, this suggests that some phylotypes of Thaumarchaeota do not assimilate intactamino acids, or carbon from amino acids at all. Together, our results demonstrate the utility of high-throughput metabolicscreening by multi-isotope nanoSIMS analysis, and underscore the importance of Thaumarchaeota in marine chemoautotrophy. Theyalso suggest intra-phylum heterogeneity in organic carbon utilization, and raise questions about the use of amino acids forcatabolic purposes by Thaumarchaeota.