The bacteriophage P1 cloning system can package and propagate DNA inserts that are up to 95 kilobases. Clones are maintained in Escherichia coli by a low-copy replicon in the P1 cloning vector and can be amplified by inducing a second replicon in the vector with isopropyl beta-D-thiogalactopyranoside. To overcome the necessity of screening clones for DNA inserts, we have developed a P1 vector with a positive selection system that is based on the properties of the sacB gene from Bacillus amyloliquefaciens. Expression of that gene kills E. coli cells that are grown in the presence of sucrose. In the new P1 vector (pAd10sacBII) sacB expression is regulated by a synthetic E. coli promoter that also contains a P1 C1 repressor binding site. A unique BamHI cloning site is located between the promoter and the sacB structural gene. Cloning DNA fragments into the BamHI site interrupts sacB expression and permits growth of plasmid-containing cells in the presence of sucrose. We have also bordered the BamHI site with unique rare-cutting restriction sites Not I, Sal I, and Sfi I and with T7 and Sp6 promoter sequences to facilitate characterization and analysis of P1 clones. We describe here the use of Not I digestion to size the cloned DNA fragments and RNA probes to identify the ends of those fragments. The positive selection P1 vector provides a 65- to 75-fold discrimination of P1 clones that contain inserts from those that do not. It therefore permits generation of genomic libraries that are much easier to use for gene isolation and genome mapping than are our previous libraries. Also, the new vector makes it feasible to generate P1 libraries from small amounts of genomic insert DNA, such as from sorted chromosomes.