We evaluated the utility of in-situ hybridization (ISH) for the rapid diagnosis of sepsis. We applied this approach to polymorphonuclear neutrophil (PMN)-rich smears from patients with suspected bacterial infection. Positive results by ISH were obtained in the smears of 123 of 292 patients (42%), while only 32 of the 292 (11%) were positive by blood culture. These findings indicate that ISH is almost four times more sensitive than the culture method for the detection of sepsis. ISH results are obtained within 1 day, while 1 day to 2 weeks is required for the results of blood culture. Blood culture and ISH methods detected the same bacteria in two patients. ISH also successfully identified the same bacteria in blood and PMN-rich body fluid (bronchoalveolar lavage samples) in 6 patients. In 19 patients, ISH of blood detected the same bacteria as those found in subcultures from other sources (e.g., stool, sputum, nasal cavity). We discuss these results in comparison with blood culture results in terms of evaluating a rapid approach to the management of patients with sepsis.