A bacterial artificial chromosome (BAC) library has been constructed for use in bovine genome mapping using the pBeloBAC11 vector. Currently, the library consists of 23, 040 clones, which achieves a 70% probability (P= 0.70) of the library containing a specific unique DNA sequence. Sixty thousand clones, or about three haploid bovine genomes, will be required to achieve a 95% probability (P= 0.95) of containing a unique sequence. An average insert size of 146 kb was estimated from the analysis of 77 randomly selected BAC clones produced by one or two rounds of size selection. The bovine DNA inserts proved to be very stable for at least 100 cell generations. No chimeric clones were detected among 11 large, size-selected BAC clones using fluorescencein situhybridization (FISH) on metaphase bovine chromosomes. The polymerase chain reaction (PCR) was used to screen the library for single-copy nuclear sequences. Thirty-three of 46 (72%) sequences were present in the library in at least one copy, which is consistent with the estimated 70% probability of this library containing a unique DNA sequence. A BAC clone containing the 3β-hydroxy-5-ene steroid dehydrogenase (HSD3B) gene was physically mapped to bovine chromosome 3 by FISH. Two new microsatellite markers were isolated from the HSD3B-positive BAC clone as sequence-tagged sites for genetic mapping. These markers cosegregated, and no recombinants were detected in 193 informative meioses. Plasmid end rescue and the inverse polymerase chain reaction methods were used to rescue both ends of this BAC clone, and chromosome walking was performed using PCR primers designed within the end region sequences. Based on our experimental results, the BAC system provides a very useful tool for complex genome analysis. © 1995 by Academic Press, Inc.