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Objectives: A new method has been developed for detecting genes determining the extended-spectrum beta-lactamase (ESBL) phenotype directly from patients' clinical material. The method enables detection of the bla(CTX-M) gene encoding CTX-M beta-lactamases and the bla(SHV) gene variants with real-time PCR technology using locked nucleic acid oligonucleotides., Material and Methods: In this pilot study, tracheal aspirates obtained from patients with mechanical ventilation hospitalized at Department of Anaesthesiology and Resuscitation of the University Hospital in Olomouc between 1st March and 30th December 2010 period were tested. Each sample was identified with standard microbiological procedures including phenotypic determination of ESBL-positive enterobacteria. At the same time, each sample was analyzed for the presence of nucleic acids (DNA) which encode CTX-M and SHV ESBL using real-time PCR., Results: 150 samples of tracheal aspirates from 71 patients were included into testing. In the set, 13 (8.7%) ESBL-positive samples were identified by culture methods while 27 (18 %) positive samples were identified by the real-time PCR method. Of the 27 PCR-positive samples, 24 were positive for the bla(CTX) gene; in 2 samples, the ESBL bla(SHV) gene was detected, and both genes were present in 1 sample. All culture-positive samples were also PCR-positive for the presence of bla(CTX) and/or bla(SHV) sequences., Conclusions: The new real-time PCR assay is likely to shorten the time for detection of enterobacteria producing SHV and CTX-M beta-lactamases from 48 to 6 hours. It enables ESBL-positive enterobacteria determination in tracheal aspirates of patients suffered from life-threatening nosocomial pneumonia where the early introduction of adequate antimicrobial treatment plays the important role.