Phylogenetic analysis of PCR-amplified 16S rRNA genes revealed the presence of archaea in picoplankton collected from the Laurentian Great Lakes in North America, Africa's Lake Victoria, and Lakes Ladoga and Onega in northeastern Eurasia. From 1 to 10% of the rRNA extracted from size-fractionated picoplankton (>0.2 μm but <1.2 μm) collected in the epilimnion and hypolimnion of these lakes was specific to the Archaea, whereas the majority of rRNA was derived from Bacteria. Analysis of the 16S rRNA genes cloned from these samples indicated they were closely related to crenarchaeal sequences that have been widely characterized from marine environments. The presence of nearly identical 16S rDNA clones in several of these geographically disparate lakes suggests a cosmopolitan distribution of specific subgroups of these Archaea in freshwater environments. Despite their abundance in the water column of freshwater lakes, we have no representatives of these crenarchaea in pure culture, and so their physiological characteristics and ecological role remain unknown.