The development of a bacteriophage P1 cloning system capable of accepting DNA fragments as large as 100 kilobase pairs (kbp) is described. The vectors used in this system contain a P1 packaging site (pac) to package vector and cloned DNA into phage particles, two P1 loxP recombination sites to cyclize the packaged DNA once it has been injected into a strain of Escherichia coli containing the P1 Cre recombinase, a kanr gene to select bacterial clones containing the cyclized DNA, a P1 plasmid replicon to stably maintain that DNA in E. coli at one copy per cell chromosome, and a lac promoter-regulated P1 lytic replicon to amplify the DNA before it is reisolated. An essential feature of the cloning system is a two-stage in vitro packaging reaction that packages vector DNA containing cloned inserts into phage particles that can deliver their DNA to E. coli with near unit efficiency. The packaging reaction can generate 10(5) clones with high molecular weight DNA inserts per microgram of vector DNA. Using NotI fragments from E. coli DNA, it was shown that the system can clone 95- and 100-kbp fragments but not a 106-kbp fragment. Presumably, the combined size of the latter fragment and the vector DNA (13 kbp) exceeds the headful capacity of P1.