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Compared with traditional PCR assays, diagnostic assays based upon real-time PCR technology have increased speed and dynamic range; in addition, they enable quantitative analysis of gene copies and have the potential for increased specificity when nucleic acid probes are used. Optimized real-time PCR assays can also be highly sensitive, detecting as few as 1-10 copies of a target gene in a nucleic acid sample. Adopting real-time PCR in a diagnostic laboratory requires an understanding of these assays, including both the benefits and drawbacks unique to this technology. An overview of real time PCR applications is presented here, with an emphasis on practical issues that might affect implementation of real-time PCR testing in a diagnostic laboratory. Increased cleanliness and process controls are required in the laboratory, to prevent contamination of sensitive real-time PCR. Nucleic acid extraction procedures, using one of the many available chemistries, should be carefully optimized for reproducible, efficient extraction of nucleic acids that are free of PCR inhibitors. Reverse transcription of RNA adds an additional variable that can affect quantitative data. For the assay itself, different options have been developed for the detection of products in real-time, including dye-based assays, hydrolysis probes, and hybridization probes. Different options and the benefits and drawbacks of each are discussed. Finally, specific applications for real-time quantitative PCR assays in diagnostic laboratories are highlighted.