We demonstrate a method for the isolation of autonomously replicating sequences from pools of clones obtained from genomic DNA libraries constructed using affinity purification of cruciform DNA. The selection of autonomously replicating sequences was based on their differential ability to replicate as episomes after transfection of pools of plasmid clones into human HeLa cells. Two separate libraries containing affinity-purified cruciform DNA were used, one prepared from DNA of log phase primary human genital fibroblasts and the other prepared from DNA of log phase SW48 colon adenocarcinoma cells. Representative samples of the entire phage libraries were converted to phagemid clones by filamentous helper phage-mediated mass excision to produce pBluescript libraries in Escherichia coli. Clones were grown up individually and the bacteria pooled into groups of 48 for recovery of plasmid DNA. Plasmid pools of 48 independent clones (120 micrograms total) were then transfected by calcium phosphate coprecipitation onto log phase HeLa cells, which were allowed to grow for 3 days before recovery of plasmid by Hirt lysis. The recovery of plasmid from each transfection was estimated to range from 10 to 60 ng. DpnI digestion was then used to digest plasmids which had not been replicated and therefore retained a bacterial methylation pattern which was sensitive to digestion. We estimated from agarose electrophoresis gels that 40-200 pg of recovered plasmid DNA per transfected pool of DNA was resistant to DpnI and therefore was capable of transforming competent E. coli cells. The DpnI-resistant fraction yielded from one to seven independent clones from each pool, with genomic DNA inserts ranging in size from 0.35 to 3.4 kb.(ABSTRACT TRUNCATED AT 250 WORDS)