The gold standard for diagnosis of classical swine fever (CSF) is cell culture virus isolation combined with reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescent antibody test (FAT) in cryosections of tonsils, spleen, various lymph nodes, ileum, and kidney. Autolytic and heterolytic samples render correct FAT evaluation difficult and can even yield false-negative or ambiguously positive results. To extend the spectrum of CSF diagnostic specimens, the authors tested whether the nictitating membrane (NM) might be a useful adjunct diagnostic specimen in wild boars and domestic pigs. To accomplish this, results of virus isolation, FAT, and RT-PCR were compared on NM samples and lymphoid tissues, which are the routine specimens of choice for CSF diagnosis. Wild boars (n = 30) and domestic pigs (n = 8) were experimentally challenged with various CSF virus (CSFV) strains or isolates of different virulence. The FAT revealed CSFV antigen in surface and tubular adenoid epithelium as well as in lymphatic follicles of the NM. In wild boars and domestic pigs with CSF, a strong agreement was found between results of FAT, virus isolation, and RT-PCR on NM and lymphoid tissues. These results suggest that NM is a useful additional specimen that can provide valuable data for postmortem diagnosis of CSF. The NM is relatively easy to sample at necropsy, and postmortem autolysis and heterolysis of this tissue is minimal compared with internal organs.