An existing canine genomic bacterial artificial chromosome (BAC) library was expanded by adding 115 200 clones with insert lengths not yet represented or under-represented. The final version of the library consists of 211 968 clones with an estimated average insert size of 110 000 base pairs. Clones were grown individually and glycerol permanents were arrayed in 2208 96-well microtitre plates. DNA of each clone was prepared by microwave treatment and organized in a three-dimensional DNA pooling system (92 superpools) allowing for rapid screening by polymerase chain reaction (PCR). Screening of the library for 111 microsatellite loci representing all canine chromosomes revealed a recovery rate of 93% and a fourfold genome coverage. In addition, 50 BAC clones containing microsatellites or genes related to ongoing projects in the laboratories of the authors and eight other research groups were successfully recovered. The present library is the first canine BAC library amenable to PCR screening and is an invaluable tool for cloning candidate loci and developing genetic markers in specific chromosomal regions. This library will also enhance mapping efforts in related canid species and is an important source for geneticists studying inherited diseases common to both dog and humans. Interested researchers can access the library following the instructions at http://www.dogmap.ch.