During the last 5 years, considerable scientific and financial efforts have been made in the development of quantitative nucleic acid detection technology. For detection of human immunodeficiency virus (HIV), quantitative culture is time consuming, cumbersome and requires appropriate laboratory safety equipment. Quantitative determination of p24 antigen by enzyme immunoassay is of limited value due to its relatively poor sensitivity. Therefore, quantitative determination of viral load using nucleic acid amplification techniques is the most accurate, prognostic marker for HIV type 1 infection, independently of the CD4+ cell count. Hepatitis B virus (HBV) is not cultivable in vitro. Serological assays allow an accurate diagnosis and follow-up of acute or chronic infection. Quantification of HBV DNA is used for the monitoring of antiviral therapy, determination of infectivity and for resolution of unclear serological profiles, e.g. isolated anti-HBc reactivity, as well as for patients in which HBV mutants are suspected. Hepatitis C virus (HCV) can only be detected by molecular based assays because no cell culture system, which permits a reliable isolation of clinical specimens, is currently available. Furthermore, early diagnosis and follow-up of infection cannot be achieved with antibody serology. The prognostic relevance of quantitative HCV RNA determination is of limited value for the long-term prognosis of chronic hepatitis C. However, viral load may predict the outcome of antiviral therapy. Genetic diversity is another challenge for HCV RNA quantification.