Sub-micrometer particles have recently been shown to exist in marine water at concentrations exceeding 10(7) particles ml-1. Their presence has important implications for ocean optics, global biogeochemical models and trophic relationships in the microbial food web. Small particles that were stainable by Acridine Orange (AO) and 4',6-diamidino-2-phenylindole (DAPI) were enumerated and sized using a quantitative fluorescence microscopy imaging system along an onshore-offshore transect from the mouth of Chesapeake Bay to the Sargasso Sea. The particles were characterized by staining with DAPI, a stain specific for double-stranded DNA and generally indicative of a living cell or viral particle, and AO, a more general bio-polymer stain indicative of organic matter. Two distinct particle populations were measured in the 0.2-1.0 mum size range: (1) typical bacteria; and (2) abundant small, dimly fluorescing (SD) particles. Surface concentrations of organic (AO-staining), SD particles ranged from 3 x 10(7) ml-1 near the mouth of Chesapeake Bay to 4 x 10(6) ml-1 in the Sargasso Sea. A variable proportion of the SD particles were DAPI-positive, probably very small bacteria and viruses. The DAPI-positive SD particles constituted 9-29% of the total organic SD particles at coastal and shelf stations, and 25-61% in a vertical profile in oligotrophic waters. The vertical distribution of SD particles in oligotrophic waters showed higher numbers in the surface layer and lower numbers below the sub-surface chlorophyll maximum, suggesting an association of the particles with biological productivity. Our carbon estimates, based on measured particle size spectra and abundances, and reasonable values for particle carbon density, agree with recent measurements of bulk elemental particulate carbon in the 0.2-0.7 mum size fraction in the Sargasso Sea. The particle volume ml-1 of the total SD particles ranged from equal to twice the bacterial biovolume ml-1, indicating a significant carbon pool.