The laboratory diagnosis of CNS infection is essential for optimal therapy. Acute infection requires rapid turn-around testing with high predictive values, that is, the ability of a test to accurately identify those patients who do or do not have disease caused by a specific etiology. The Gram's stain, fungal stains of direct smears, antigen testing for C. neoformans, and culture of bacteria, fungi, mycobacteria, and some viruses are important tests for the diagnosis of acute infection. The laboratory diagnosis of chronic infection necessitates discussion between the clinician and laboratory technician to allow triaging of testing. Antigen tests for bacteria, fungi, and viruses; antibody tests for multiple microorganisms; and PCR testing for bacteria, M. tuberculosis, and many viruses are all important in limited clinical situations. All testing for acute or chronic disease depends on sufficient specimen that is transported to the laboratory in a manner that will not compromise viability or chemical integrity. Sterile containers that maintain moisture content, exclude oxygen for anaerobic requests, and are stored at proper temperatures (22 degrees C room, 4 degrees C refrigeration, or -20 degrees C freezer depending on pathogen and test) are mandatory. Many laboratory issues addressing the diagnosis of CNS infection are changing or evolving. Most important is the recognition that bacterial antigen testing for the diagnosis of acute bacterial meningitis rarely impacts patient management and is not routinely needed, CSF shunt infections differ from usual meningeal infections and require rapid diagnosis, and TB meningitis remains a difficult disease to diagnosis but may be confirmed first by PCR testing of CSF. In addition, Whipple's disease of the CNS can be confirmed using PCR with CSF; CJD has a marker protein, referred to as 14-3-3 antigen, that can be detected in CSF, and the diagnosis of fungal CNS disease requires careful interpretation of direct smears, antigen and antibody testing, and culture. Most difficult to diagnose among the CNS infections are viral meningitis and encephalitis. The appearance of new etiologies, such as West Nile virus, and the common use of PCR for the herpes viruses and enteroviruses represent important advances. Evolving methods for the laboratory diagnosis of CNS infection represent significant improvements over previous testing; however, the array of tests available demands more attention for appropriate selection, is significantly more expensive, and requires new skills for performance and interpretation. The responsibility for proper use of laboratory testing lies both with the clinician and laboratory technician.