Recovery of Streptococcus pneumoniae from positive blood culture bottles may be difficult due to autolysis of pneumococci. Therefore, we evaluated the performance of the Binax NOW S. pneumoniae antigen test with samples from positive blood culture bottles and defined the duration of detectable pneumococcal antigen in these bottles. Use of the S. pneumoniae antigen test is an alternative method for identifying S. pneumoniae from positive blood culture bottles and may enable a diagnosis of pneumococcal bacteremia despite negative subcultures.