In order to understand how environmental factors shape the diversity of Prochlorococcus in the Atlantic Ocean, we have elucidated the microdiversity along a north-south transect. The polymerase chain reaction-restriction fragment length polymorphism analysis of the genetic diversity of rpoC1 gene fragments of Prochlorococcus at 12 sampling sites revealed a latitudinal pattern in Prochlorococcus RFLP-type diversity in the samples collected from two depths. At the depth to which 14% of surface irradiance penetrated, HLII clones dominated the stations closest to the equator. The percentage of HLI clones increased with distance from the equator and LL clones were found only at the most northern and southern stations. In contrast, deeper (1% light depth) water samples did not show any overall trend in Prochlorococcus diversity or clade dominance. Multivariate statistical analyses indicated that Prochlorococcus diversity was linked to water temperature (partially an effect of latitude) and depth (which was linked to light penetration and turbidity). Phylogenetic analysis of the sequences obtained from the 423 different environmental RFLP-types detected in this study indicated that the HLII and HLI populations were composed of a wide range of genetically different clones, while the LL Prochlorococcus clade was less diverse, although half of the samples screened in this study derived from the 1% light depth.