In order to determine whether a blood culture positive for coagulase-negative staphylococci (CNS) represents bacteremia or contamination, a prospective study was conducted using molecular typing to analyze CNS blood culture isolates and corresponding CNS skin isolates collected after skin disinfection from 431 subjects. CNS bacteremia was not found in any of the 301 subjects not suspected of having bacteremia. In 130 patients suspected of having bacteremia, the rate of actual CNS bacteremia was 6%. The overall rate of CNS blood culture contamination was 1%. Chart analysis showed good agreement between our microbiological definitions of bacteremia and the clinical definitions previously published. Bacteremia and contamination can be differentiated using pulsed-field gel electrophoresis and molecular typing of CNS isolates obtained from cultures of blood and corresponding skin samples.