Porcine picornaviruses comprising at least 23 serotypes grouped into six species were described as causative agents of neurological disorders, reproductive failure, and aphthae-like dermal lesions of swine. Other viruses such as classical swine fever virus (CSFV), African swine fever virus, pseudorabies virus (PRV), vesicular stomatitis virus, vesicular exanthema virus, porcine respiratory and reproductive syndrome virus, and porcine parvovirus (PPV) may cause diseases with similar clinical symptoms. Therefore, rapid and reliable PCR detection of the most frequent porcine picornaviruses is of interest. A real-time RT-PCR protocol employing LightCycler technology to detect all known serotypes of the three porcine enterovirus (PEV) cytopathic effect (CPE) groups was established. It uses three sets of primer pairs and group-specific hybridisation probes. The primer pairs were designed to amplify highly conserved sequences of the 5'-non-translated region (5'-NTR) of the relevant virus species. The one-step real-time PCR based on the LightCycler technology is more rapid and less contamination-prone than the nested RT-PCR and allows the precise quantitation of the virus load in the tested specimens. All acknowledged serotypes of the three PEV CPE groups and all tested field strains isolated from clinical specimens were detectable. Viruses of the PEV CPE group III can be distinguished from the closely related swine vesicular disease virus (SVDV).