Mitochondrial DNA from 49 Lipizzan horses representing 16 maternal lines from the original stud at Lipica was used for SSCP analysis and DNA sequencing. The SSCP analysis of the 444 bp long fragment of the D-loop region extending from the tRNA(Pro) gene to the central conserved sequence block revealed three distinct groups of SSCP patterns. Both ends of the D-loop region (378 bp and 310 bp), which are considered as the most variable regions within the mammalian mitochondrial DNA, were sequenced. According to 49 polymorphic sites identified within the both parts of the D-loop region, the 16 maternal lines were grouped into 13 distinct mitochondrial haplotypes. The minimal difference between two different haplotype DNA sequences was one nucleotide and the maximal 24 nucleotides. The inheritance of mitochondrial haplotypes was stable and no sequence variation potentially attributable to mutation within maternal line was observed. Considerable DNA sequence similarity of Lipizzan mitochondrial haplotypes with the haplotypes from other breeds was observed. Phylogenetic analysis of the sequence data revealed a dendrogram with three separated branches, supporting the historical data about the multiple origin of the Lipizzan breed.