A bovine bacterial artificial chromosome (BAC) library was screened for the presence of eight type I anchor loci previously used within hybrid somatic cells and an interspecies hybrid backcross to construct a genome map of bovine chromosome 19 (BTA19). Six out of eight loci were identified in the BAC library (NF1, CRYB1, CHRNB1, TP53, GH1 and P4HB). The BACs were then used in single-colour fluorescence in situ hybridization (FISH) to assign these genes to BTA19 band locations. Gene order was determined by single-colour FISH, and was confirmed by dual-colour FISH to mitotic and meiotic chromosomes. The order, centromere-NF1-CRYB1-CHRNB1-TP53-GH1-P4HB, was in agreement with the order determined by linkage analyses. In addition, the order of CHRNB1 and TP53, previously unresolved by linkage analyses, was established. These data provide high-resolution cytogenetic anchorage of the BTA19 genome map from chromosome bands 14-22.