Bloodstream infections (BSI) are an important cause of morbidity and mortality. Increasing rates of antimicrobial-resistant pathogens limit treatment options, prompting an empirical use of broad-range antibiotics. Fast and reliable diagnostic tools are needed to provide adequate therapy in a timely manner and to enable a de-escalation of treatment. The Accelerate Pheno system (Accelerate Diagnostics, USA) is a fully automated test system that performs both identification and antimicrobial susceptibility testing (AST) directly from positive blood cultures within approximately 7 h. In total, 115 episodes of BSI with Gram-negative bacteria were included in our study and compared to conventional culture-based methods. The Accelerate Pheno system correctly identified 88.7% (102 of 115) of all BSI episodes and 97.1% (102 of 105) of isolates that are covered by the system's identification panel. The Accelerate Pheno system generated an AST result for 91.3% (95 of 104) samples in which the Accelerate Pheno system identified a Gram-negative pathogen. The overall category agreement between the Accelerate Pheno system and culture-based AST was 96.4%, the rates for minor discrepancies 1.4%, major discrepancies 2.3%, and very major discrepancies 1.0%. Of note, ceftriaxone, piperacillintazobactam, and carbapenem resistance was correctly detected in blood culture specimens with extended-spectrum beta-lactamase-producing Escherichia coli (n ϭ 7) and multidrug-resistant Pseudomonas aeruginosa (n ϭ 3) strains. The utilization of the Accelerate Pheno system reduced the time to result for identification by 27.49 h (P Ͻ 0.0001) and for AST by 40.39 h (P Ͻ 0.0001) compared to culture-based methods in our laboratory setting. In conclusion, the Accelerate Pheno system provided fast, reliable results while significantly improving turnaround time in blood culture diagnostics of Gramnegative BSI.KEYWORDS antimicrobial susceptibility testing, bloodstream infections, rapid tests B loodstream infections (BSI) are an important cause of morbidity and mortality, associated with prolonged hospital stays, as well as high costs for health care systems (1-3). Increasing rates of antimicrobial-resistant pathogens, in particular Gramnegative bacteria, limit treatment options, often prompting the empirical use of broad-range antibiotics. Therefore, we need fast and reliable diagnostic tools to speed up both identification (ID) and antimicrobial susceptibility testing (AST) from blood cultures in order to provide adequate therapy in a timely manner and to enable effective antibiotic stewardship interventions (4).