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A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14 % of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheteroge-neity typical of sequence clusters detected in environmental clone libraries. The use of PCR targeting the 16S rRNA gene has provided a powerful, culture-independent means of characterizing mi-crobial communities, which has advanced our understanding o