Microarrays are useful tools for detecting and quantifying specific functional and phylogenetic genes in natural microbial communities. In order to track uncultivated microbial genotypes and their close relatives in an environmental context, we designed and implemented a 'genome-proxy' microarray that targets microbial genome fragments recovered directly from the environment. Fragments consisted of sequenced clones from large-insert genomic libraries from microbial communities in Monterey Bay, the Hawaii Ocean Time-series station ALOHA, and Antarctic coastal waters. In a prototype array, we designed probe sets to 13 of the sequenced genome fragments and to genomic regions of the cultivated cyanobacterium Prochlorococcus MED4. Each probe set consisted of multiple 70-mers, each targeting an individual open reading frame, and distributed along each approximately 40-160 kbp contiguous genomic region. The targeted organisms or clones, and close relatives, were hybridized to the array both as pure DNA mixtures and as additions of cells to a background of coastal seawater. This prototype array correctly identified the presence or absence of the target organisms and their relatives in laboratory mixes, with negligible cross-hybridization to organisms having <or= approximately 75% genomic identity. In addition, the array correctly identified target cells added to a background of environmental DNA, with a limit of detection of approximately 0.1% of the community, corresponding to approximately 10(3) cells ml(-1) in these samples. Signal correlated to cell concentration with an R(2) of 1.0 across six orders of magnitude. In addition, the array could track a related strain (at 86% genomic identity to that targeted) with a linearity of R(2) = 0.9999 and a limit of detection of approximately 1% of the community. Closely related genotypes were distinguishable by differing hybridization patterns across each probe set. This array's multiple-probe, 'genome-proxy' approach and consequent ability to track both target genotypes and their close relatives is important for the array's environmental application given the recent discoveries of considerable intrapopulation diversity within marine microbial communities.