Nitrogen (N 2 ) fixation is a globally important process often mediated by diazotrophic cyanobacteria in the open ocean. In 2010, seawater was collected near Cape Verde to identify and measure N 2 and carbon (C) fixation by unicellular diazotrophic cyanobacteria. The nifH gene abundance (10 4 –10 6 nifHL −1 ) and nifH gene transcript abundance (10 2 –10 4 cDNAnifHL −1 ) for two unicellular groups, UCYN-A and UCYN-B, were detected. UCYN-A was also identified and quantified (10 4 –10 5 cellsL −1 ) by new probes (UCYN-A732 and UCYN-A159) using Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) assays. The UCYN-A were observed as free cells or attached to a larger unidentified eukaryotic cell. A Halogen In Situ Hybridization-Secondary Ion Mass Spectrometry (HISH-SIMS) assay using the UCYN-A732 probe was applied on samples previously incubated with 13 C-bicarbonate and 15 N 2 . Free UCYN-A cells were enriched in both 13 C and 15 N and estimated C and N 2 fixation rates for UCYN-A were lower compared to co-occurring unicellular cyanobacteria cells similar in size (3.1–5.6μm) and pigmentation to diazotroph Crocosphaera watsonii. Here, we identify and quantify two common co-occurring unicellular groups and measure their cellular activities by nanoSIMS.