The displacement-loop (D-loop) region of bovine mitochondrial DNA (mtDNA) isolated from blood leukocytes was studied. A method was devised for the isolation of mtDNA from blood leukocytes based on detergent lysis of the plasma membrane and removal of cell nuclei by centrifugation. Nucleic acids extracted from the residual cytoplasmic fraction contained mtDNA in quantities sufficient for restriction enzyme analysis and for DNA cloning and sequencing;MtDNA was from a herd of Holstein cattle that was established 1968 from diverse genetic sources. Maternal lineages were traced by pedigree analysis to their earliest origins in the late 1800s. MtDNA was isolated from blood leukocytes and nucleotide sequences were determined for the light strand of the 910 base-pair (bp) D-loop region. Fifty-one sites of sequence variation in 38 maternal lineages were identified. Nucleotide substitutions were found at 48 sites (38 transitions and 10 transversions); one nine-bp deletion, and two variable-length poly G/poly C regions of 6-8 bp and of 11-16 bp were found. Nucleotide substitutions were distributed nonrandomly across the D-loop region with avoidance of the 5[superscript]' end (100 bp), a central GC-rich region (80 bp), and the 3[superscript]' end that contains light and heavy strand transcriptional promoters and the origin of heavy strand replication. Variation within maternal lineages was observed at two sites: One G-to-C transversion at nucleotide (nt) 363 and in the length of a poly G/poly C region that extends from nt 351 to nt 363. A cytoplasmic gene tree constructed from the sequence data revealed a bifurcation in the mtDNA of cattle into two major groups based on a transition at nt 169. Sufficient variation seems to exist in mtDNA of cattle for the assignment of animals to cytoplasmic lineages.