In large areas of the world's oceans, biological dinitrogen (N2) fixation supports a significant fraction of ecosystem productivity; to date, however, there is little information on the abundances of specific diazotrophs in the ocean. In this study, the vertical distributions of several different groups of N2-fixing bacteria were examined using quantitative polymerase chain reaction (QPCR) amplification of group-specific dinitrogenase reductase (nifH) genes from Stn ALOHA in the subtropical North Pacific Ocean. Depth distributions (<200 m) of 3 cyanobacterial nifH phylotypes and 1 previously uncharacterized Cluster III nifH phylotype were evaluated. The nifH-containing cyanobacteria included sequence-types similar to Trichodesmium spp. and 2 nifH sequences closely related to unicellular cyanobacteria (termed Groups A and B). The Group A cyanobacteria (most closely related to Cyanothece sp.) were the most abundant of all phylotypes examined, comprising 2 × 105 nifH gene copies l-1 in the high-irradiance (>700 μmol quanta m-2 s-1), nitrate-depleted (<10 nmol l-1) upper-ocean waters. Group B cyanobacterial phylotypes (most closely related to Crocosphaera watsonii) demonstrated a depth distribution similar to Group A, but Group B nifH abundance was considerably lower, averaging 2 × 103 nifH gene copies l-1 in the upper photic zone. The abundance of Trichodesmium spp. ranged from 1 × 103 to 7 × 103 nifH gene copies l-1 in the upper ocean, declining to <100 nifH gene copies l-1 below the mixed layer (∼82 m). The Cluster III nifH phylotype was the most abundant nifH phylotype in the dimly lit (<12 μmol quanta m-2 s-1) lower photic zone (>100 m). These results revealed differences in the depth distributions of N2-fixing plankton at Stn ALOHA, and suggest that unicellular diazotrophs comprise a significant component of plankton biomass in this oligotrophic marine ecosystem.