ABSTRACT: The practical value of blood cultures in the diagnosis of sepsis is impaired by a delay in the turnaround time to result and by the fact that blood culture positive can be found for only about 30% of these patients. Conventional laboratory signs of sepsis and acute phase protein biomarkers are sensitive and easy to use, but often also very nonspecific. Molecular diagnostic reflects currently the most promising avenue to decrease time to result and to influence decision making for antibiotic therapy in the septic host. In this study, we wish to highlight the impact of the LightCycler SeptiFast, a multipathogen probe-based real-time polymerase chain reaction, in the rapid etiological diagnosis of sepsis in patients with clinical and laboratory signs of bloodstream infections. We have evaluated prospectively 830 adult patients with suspected bloodstream infection and at least two criteria of systemic inflammatory response syndrome. In more than 50% of critically ill patients strongly suspected of having sepsis, we arrived to an etiological diagnosis only by the molecular method in a median time of 15 h, with specificity and predictive positive values of 96% and 94%, respectively. We highlight the role of DNAemia as time-critical, high-specificity, etiological, non–culture-based rule-in diagnostic biomarker in patients with presumed sepsis.