During a study on Bovine Viral Diarrhoea (BVD) epidemiology in Thailand, a pestivirus was detected in serum from a calf. Comparative nucleotide sequence analysis showed that this virus was closely related to a recently described atypical pestivirus (D32/00_'HoBi') that was first isolated from a batch of foetal calf serum collected in Brazil. The results from virus neutralisation tests performed on sera collected from cattle in the herd of the infected calf, showed that these cattle had markedly higher antibody titres against the atypical pestivirus 'HoBi' than against Bovine Viral Diarrhoea Virus types 1 and 2, or Border Disease Virus. The results also supported, consequently, the results from the molecular analysis, and demonstrated that a 'HoBi'-like pestivirus had been introduced to, and was now circulating in the herd. This study is the first to report a natural infection in cattle with a virus related to this atypical pestivirus, and it suggests that this group of pestiviruses may already be spread in cattle populations. The findings have implications for BVD control and for the biosafety of vaccines and other biological products produced with foetal calf serum. Consequently, these atypical pestiviruses should be included in serological assays, and any diagnostic assay aimed at detection of pestiviruses in biological products or animals should be tested for its ability to detect them.