A bovine genomic large‐insert bacterial artificial chromosome (BAC) library has been constructed from leukocytes of a Holstein‐Friesian male. Size fractionated __Dpn__II‐digested genomic DNA was ligated to the dephosphorylated __Bam__H1 ends of a pBACe3·6 vector. Approximately 8·3×104 individual BAC clones were picked into 384‐well plates. Two‐hundred and sixty‐seven randomly chosen clones were characterized by pulsed‐field gel electrophoresis (PFGE). The average insert size was 104 kb with a frequency of clones without inserts of 5·5%. Thirty‐four BAC clones were mapped by fluorescence in situ hybridization (FISH) to cattle chromosomes. Three showed signals at more than one location, one of them on the centromeric regions of all autosomes, indicating that the clone contains centromeric repeats. A subset of these BAC clones was used for the development of sequence tagged sites. Both subcloning and direct sequencing of the BACs were used for generating sequence tagged site information. The clones from the library were gridded onto high‐density membranes, and PCR superpools were produced from the same set of clones. Membranes and superpools are available through the Resource Centre of the German Human Genome Project in Berlin (http://www.rzpd.de).