There were 2030 designated cases of foot-and-mouth disease (FMD) during the course of the epidemic in the UK in 2001 (including four from Northern Ireland). Samples from 1720 of the infected premises (IPs) were received in the laboratory and examined for either the presence of FMD virus (virological samples from 1421 IPs) or both FMD virus and antibody (virological and serological samples from 255 IPs) or antibody alone (from 44 IPs). The time taken to issue final diagnostic results ranged from a few hours in cases in which positive results were obtained by ELISA on epithelia containing sufficient virus to be detected, to several days for samples containing small amounts of virus requiring amplification through cell culture, negative samples or samples tested for antibody. Two subsets of samples were analysed retrospectively by real-time reverse transcriptase-PCR (RT-PCR); first, epithelia that were negative by both ELISA and virus isolation (VI) in cell culture, and secondly, samples that were negative by ELISA on epithelial suspension but positive by VI. There was broad agreement between the RT-PCR and VI/ELISA combined, except that the RT-PCR procedure did not detect a group of related virus isolates from Wales. These viruses had evidently evolved during the epidemic and had a nucleotide substitution in the RT-PCR probe site, which prevented them from being detected by the routine diagnostic probe. No evidence of FMD virus, antibody or nucleic acid was found in approximately 23 per cent (390 of 1730) of IPs from which samples were received, suggesting that the incidence of FMD during the outbreak may have been over-reported.