Streptococcus pneumoniae is one of the most common pathogens in bacterial meningitis. Rapid diagnosis is critical for effective treatment. The aim of this study was to assess the accuracy of the NOW S. pneumoniae Urinary Antigen Test, (Binax, Portland, ME, USA) originally developed for urine testing, in detecting the S. pneumoniae antigen in cerebrospinal fluid (CSF). The study included 519 patients with suspected meningitis. CSF, blood and urine samples were cultured according to standard methods. CSF viral culture was also performed. CSF and urine specimens were tested for pneumococcal antigen with the NOW S. pneumoniae test.S. pneumoniae was isolated from the CSF of 22 patients. The direct antigen test was positive in CSF in 21/22 patients (95.4% sensitivity), and in urine, in 12/21 (57.1% sensitivity). Direct CSF smear was positive in 15/22 (68% sensitivity). CSF samples that cultured negative for S. pneumoniae (n = 470) or positive for other bacteria (n = 27) were also negative on the NOW test (100% specificity). By contrast, urine samples of 63/470 of patients with negative CSF culture were positive on the NOW test, as were 5/27 urine samples of patients with CSF culture positive for other bacteria (p = 0.45). The NOW S. pneumoniae antigen test in CSF yields a rapid and very reliable diagnosis of pneumococcal meningitis, enabling prompt and adequate treatment. Its low sensitivity in urine indicates that this mode of testing is not useful for the diagnosis of pneumococcal meningitis. These data have been included in the FDA application for approval of the NOW test for use in the CSF for the diagnosis of pneumococcal meningitis.