A new subspecies, Staphylococcus horninis subsp. novobiosepticus, isolated from human blood cultures, a wound, a breast abscess and a catheter tip, is described on the basis of a study of 26 strains isolated between 1989 and 1996.DNA-DNA reassociation reactions, conducted under stringent conditions, and macrorestriction pattern analysis demonstrated that these strains are closely related to previously characterized 5. horninis strains isolated from human skin and clinical specimens, but are significantly divergent. 5. horninis subsp. novobiosepticus can be distinguished from 5. horninis (now named 5. horninis subsp. horninis) by its combined characteristics of novobiocin resistance and failure to produce acid aerobically from D-trehalose and N-aCety1-Dglucosamine. Furthermore, all 26 strains of the new subspecies are resistant to nalidixic acid, penicillin G, oxacillin, kanamycin and streptomycin, and were either resistant or had intermediate resistance to methicillin and gentamicin. Most strains were also resistant to erythromycin, clindamycin, chloramphenicol, trimethoprim/sulfamethoxazole and ciprof loxacin. Based on a comparison of the sequences of a 1001 bp rned amplification product from reference methicillin-resistant staphylococci, the rned gene present in S-horninis subsp. novobiosepticus was identified as homologue A, commonly found in S. aureus and many coagulase-negative staphylococcal species. The type strain of 5. horninis subsp. novobiosepticus is ATCC 700236l. Descriptions of S. horninis subsp. novobiosepticus subsp. nov. and 5. horninis subsp. horninis are given and the description of S. horninis is emended.