In the present study 130 S. uberis strains and one S. parauberis strain isolated from bovine milk samples of 58 different farms of various locations in Hesse, Germany, as well as two reference strains of each species were comparatively investigated for cultural, biochemical, serological and molecular properties. All S. uberis strains produced the enzyme beta-D-glucuronidase, while the S. parauberis strains were negative. The S. uberis and S. parauberis 16S rRNA genes were amplified by polymerase chain reaction and subsequently digested with the restriction enzymes RsaI and AvaII yielding species-specific restriction patterns. Both species were additionally identified by amplifying species-specific parts of the genes encoding the 16S rRNA, the 23S rRNA and the 16S-23S rDNA intergenic spacer region, respectively. The CAMP factor gene cfu, a potential virulence factor of S. uberis, was amplified, corresponding to a phenotypically positive CAMP-reaction, using cfu-specific oligonucleotide primers. In addition the streptokinase/plasminogen activator encoding genes skc/pauA, a second potential virulence factor, could be amplified for 126 of the 130 S. uberis but not for S. parauberis. A DNA fingerprinting of S. uberis strains, performed by macrorestriction analysis of their chromosomal DNA by pulsed-field gel electrophoresis, revealed that most of the isolates were not related to each other. However, identical DNA patterns were noted for some of the isolates within different quarters of an individual cow and also for different cows within the same farm. The generally unrelated DNA patterns indicated that S. uberis is a pathogen with multiple environmental habitats and that infections are caused by a great variety of strains.