Infections with human enteroviruses are common worldwide and cause a wide range of signs and symptoms. Nowadays in current diagnostics procedures older virological methods, such virus isolation in a cell cultures and seroneutralisation assay, are replaced with molecular biology tests. The aim of the study was development of real-time PCR assay for detection of human adenoviruses. DNA isolated from MK2 cell line infected with nineteen different enterovirus strains was used for development of a qualitative real-time PCR assay using primers targeting a conserved region of the 5'UTR region and a specific TaqMan probe. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of Coxackie A9 cDNA in range between 10 degrees and 10(-8). For comparison typical end-point detected RT-PCR for enterovirus detection with the same cDNA dilutions was made. The sensitivity of novel method was about ten thousand-fold higher than older one. The conclusion is that real-time PCR is very advisable in diagnostics of diseases caused with enteroviruses. The high level of sensitivity, specificity, accuracy, and rapidity provided by this assay are favorable for the use in the detection of enteroviral RNA in clinical specimens, especially from neuroinfections.