A working scheme developed in our laboratory for identification (by species group and species) of coagulasenegative staphylococci (CNS) was evaluated with 201 consecutive isolates and then validated by using the reference method of Kloos and Schleifer (W. E. Kloos and K. H. Schleifer, J. Clin. Microbiol. 1: [82][83][84][85][86][87][88] 1975). This five-test simple scheme (referred to here as the simple scheme) combines the novobiocin susceptibility test with tests for urease, pyrrolidonyl arylamidase, ornithine decarboxylase, and aerobic acid from mannose. The addition of one or two tests within a particular species group could then positively identify the isolate. Two commercial systems, Staph-Zym (Rosco) and API-Staph (bioMérieux), along with results obtained by using Rosco diagnostic tablets (nongrowth tests), were also compared with the reference method. One isolate could not be identified even by the reference method. Of the remaining 200 strains, 191 (95.5%) strains were correctly identified with Staph-Zym and 171 strains (85.5%) were correctly identified with API-Staph. The most frequent clinical CNS species isolated were Staphylococcus epidermidis (50.5%), S. haemolyticus (18.5%), S. saprophyticus subsp. saprophyticus (16.0%), S. lugdunensis (6.0%), and S. warneri (2.5%). The simple scheme validated with the reference method has demonstrated an excellent correlation in the identification of the three most frequent species isolated: S. epidermidis, S. haemolyticus, and S. saprophyticus subsp. saprophyticus. With the simple scheme, identification of CNS was possible within 24 h after the enzymatic tests were used, whereas up to 72 h is necessary for the growth tests. This methodology would be very useful in any clinical microbiology laboratory for the presumptive identification of CNS species groups and species.Many studies have been initiated since 1958 as a result of the growing recognition that coagulase-negative staphylococci (CNS) are clinically important (28) in an attempt to classify these organisms (1, 21). In the 1970s, W. E. Kloos and K. H. Schleifer determined the natural relationships of CNS based on systematic studies that allowed these researchers to resolve and characterize different CNS species (14). Species were identified based on an ensemble of morphological, physiological, and biochemical characteristics, antibiotic susceptibility patterns, and cell wall composition.In the last decade, molecular studies contributed to a notable progress in the classification of staphylococci and in the development of methods for identifying them at the genus, species, subspecies, and strain levels (5). Although Staphylococcus epidermidis accounts for most CNS infections, many other species have been identified in association with human infections (3, 27, 29). The importance of CNS as major nosocomial pathogens is mainly associated with prosthetic and indwelling devices such as prosthetic joints, heart valves, pacemaker implants, ventricular-peritoneal shunts, and peritoneal dialysis catheters and with s...