Purpose: The discrimination between infectious and non-infectious causes of acute respiratory failure (ARF) in hospitalized patients admitted to the intensive care unit (ICU) is difficult. Using a novel diagnostic test measuring the expression of four RNA biomarkers in blood (SeptiCyte LAB) we aimed to distinguish between infection and inflammation in this setting. Methods: We enrolled hospitalized patients with ARF requiring prompt intubation in the ICU from 2011 to 2013. We excluded patients having an established infection diagnosis or an evidently non-infectious reason for intubation. Blood samples were collected upon ICU admission. Test results were categorized into four probability bands (with higher bands indicating a higher probability of infection) and compared with the plausibility of infection as rated by post-hoc assessment using predefined definitions. Results: Of 467 included patients, 373 (80%) were treated for a suspected infection at admission. Plausibility of infection was classified as ruled-out, undetermined, or confirmed in 41 (11%), 135 (36%), and 197 (53%) of these, respectively. Overall, the pre-test probability of infection was 42%. Test results correlated with the plausibility of infection (Spearman′s rho 0.332; p<0.001). After exclusion of undetermined cases, positive predictive values were 29%, 54%, and 76% for probability bands 2, 3, and 4, respectively, whereas the negative predictive value for band 1 was 76%. However, SeptiCyte LAB did not outperform CRP when comparing diagnostic discrimination (AUC 0.731; 95%CI 0.677-0.786 vs. 0.727; 95%CI 0.666-0.788). Conclusion: In a setting of hospitalized patients admitted to the ICU with ARF, the diagnostic value of SeptiCyte LAB seems limited.