In November 2004, Chaetoceros spp. (diatom) cells were collected from 5 m at Station ALOHA (22º45′ N, 158º0′ W) in the subtropical North Pacific Ocean. Attached to the spines of several Chaetoceros spp. were symbiotic heterocystous cyanobacterial cells, identified as Calothrix rhizosoleniae Lemmerm. The symbiotic diatom cells were handpicked and placed in N-deplete media. The host cells have been absent since isolation, and the cyanobacterial symbiont has persisted in a free-living state in two different liquid and solid media: one made from artificial seawater, and another with natural seawater. All media are devoid of fixed inorganic nitrogen. Trichome length varied from three to four vegetative cells when growing colonially, to >10 vegetative cells when growing as single trichomes. The DNA and amino acid sequences of the narB, rbcL, and rnpB genes are most similar to those from other heterocystous cyanobacteria (Anabaena and Fischerella). Acetylene reduction (AR) assays were run in conjunction with multiplexing quantitative reverse transcription–PCR (qRT–PCR) assays. Gene transcription for rbcL and nifH was high, coincident with maximum AR, and occurred in the middle of the photoperiod. Eight irradiance curves of nitrogenase activity at varying biomass concentrations showed evidence of photoinhibition at high light intensities. Here, we report on the genetic identification and photophysiology for the first symbiotic isolate, Ca. rhizosoleniae SC01, of an open-ocean diatom (Chaetoceros).