An investigation into the genetic structure of Prochlorococcus picophytoplankton in depth profiles from the Sargasso Sea and the Gulf Stream revealed a high degree of genetic heterogeneity within local populations. partial sequencing of cloned polymerase chain reaction products amplified from flow cytometrically sorted cells was used to identify 6-21 different Prochlorococcus petB/D alleles recovered from each of eight sorted samples, with 68 alleles identified among 187 clones in the combined data set. Rarefaction analyses indicated that many additional unsampled alleles were present at each level of sample aggregation. Overlapping sets of alleles were recovered from Prochlorococcus populations in the two water columns, from different depths within each water column, and from flow cytometrically distinguishable subpopulations sorted from the same water sample, suggesting that each of these populations drew their membership from a single gene pool. Consistent with results of autecological studies, members of a high-light-adapted Prochlorococcus clade predominated in clone libraries from surface waters. It thus appears that wild Prochlorococcus populations consist of individuals drawn from a variety of evolutionary Lineages and that populations at different depths and in two distinct hydrographic regimes recruit their members from the same gene pool. Natural selection favors the predominance of high-light-adapted genotypes in near-surface populations drawn from this gene pool.