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Phytoplankton communities residing in the open ocean, the largest habitat on Earth, play a key role in global primary production. Through their influence on nutrient supply to the euphotic zone, open-ocean eddies impact the magnitude of primary production and its spatial and temporal distributions. It is important to gain a deeper understanding of the microbial ecology of marine ecosystems under the influence of eddy physics with the aid of advanced technologies. In March and April 2018, we deployed autonomous underwater and surface vehicles in a cyclonic eddy in the North Pacific Subtropical Gyre to investigate the variability of the microbial community in the deep chlorophyll maximum (DCM) layer. One long-range autonomous underwater vehicle (LRAUV) carrying a third-generation Environmental Sample Processor (3G-ESP) autonomously tracked and sampled the DCM layer for four days without surfacing. The sampling LRAUV’s vertical position in the DCM layer was maintained by locking onto the isotherm corresponding to the chlorophyll peak. The vehicle ran on tight circles while drifting with the eddy current. This mode of operation enabled a quasi-Lagrangian time series focused on sampling the temporal variation of the DCM population. A companion LRAUV surveyed a cylindrical volume around the sampling LRAUV to monitor spatial and temporal variation in contextual water column properties. The simultaneous sampling and mapping enabled observation of DCM microbial community in its natural frame of reference.