Infectious diseases are common in patients admitted to the intensive care unit (ICU). Microbial culture and serology are traditionally used to establish the diagnosis, but take considerable time and may provide false-negative results after use of antibiotics or in the early phase of infection. Antigen detection, fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) are increasingly used as alternatives for culture and serology in the routine diagnostic procedures of the microbiological laboratory. Advantages of these techniques are the short turnaround time to results, the higher sensitivity, and that these tests are presumably not affected by prior use of antibiotics. In addition they have a higher yield for detection of fastidious microorganisms and are very suitable for detection of one specific microorganism in samples with a high diversity of microbes. A disadvantage is that false-positive results may occur due to colonization or laboratory contamination. Furthermore, antimicrobial susceptibility data usually cannot be generated with these molecular techniques. This review discusses the pros and cons of these commonly used laboratory techniques for rapid diagnosis of infection in ICU patients. Copyright © 2008, Nederlandse Vereniging voor Intensive Care. All rights reserved.