For a comparative characterization of the lungworm species D. viviparus and D. eckerti which is not generally accepted as a separate species, the restriction fragment length polymorphism (RFLP) of the PCR amplified ribosomal second internal transcribed spacer (ITS2) and their sequences of both species have been examined. Ribosomal ITS2 DNA was amplified from genomic DNA of individual worms using primer that correspond to the conserved 3' and 5' ends of the ITS2 flanking 5.8S and 28S regions of Caenorhabditis elegans. PCR products were digested with restriction endonucleases AluI, NspI, SspI, BclI and MseI and separated electrophoretically on a 1% agarose gel. Each restriction enzyme produced a species specific fragment length pattern. PCR products were cloned into pCRII and sequenced. The length of the ITS2 varied between 403 (D. viviparus) and 481 bases (D. eckerti) with a GC content ranged from 25 to 33%. Intraspecific variations were low (0-1.5%). Interspecific differences occur at 112 bases. The sequence homology between D. viviparus and D. eckerti was found with 76.7%. ITS2 sequence differences between D. viviparus and D. eckerti by far exceeded intraspecific variations. Therefore both methods showed distinct differences between the lungworm species examined, thus proving that D. eckerti is correctly described as a separate species. Both species occur in deer and specific primers have been designed for both species that will be used in prevalence studies to investigate the actual role of deer in the transmission of D. viviparus to cattle.