Foot-and-mouth disease is a highly contagious disease of cloven hooved animals. In cattle, both acute and long-term persistent infections occur. Foot-and-mouth disease virus (FMDV), a picornavirus, has been shown, using virus isolation procedures, to replicate in the pharynx and soft palate of cattle. In this study, in situ hybridization has been used to detect FMDV RNA within the cells of tissues removed from infected bovines. A digoxigenin-labelled anti-sense RNA probe was prepared corresponding to a region of the FMDV genome encoding part of the RNA-dependent RNA polymerase (3D). The efficacy and specificity of this probe for in situ hybridisation was determined using virus-infected cells in tissue culture. Strong cytoplasmic staining was only detected in FMDV-infected cells. Various tissue samples were collected from FMDV-infected cattle between 5 and 17 days post-infection. Viral RNA was detected by in situ hybridisation within cells of the soft palate, tonsil and pharynx up to 17 days post-infection. This technique is useful for the study of FMDV localization in cattle both during and after the acute clinical phase of disease and may assist in identifying specific sites of virus persistence. Copyright (C) 1999 Elsevier Science B.V.