Total DNA extracted from mucosal and skin lesions of captive and stranded cetaceans was analyzed for herpesvirus DNA by nested and direct polymerase chain reactions (PCR). The targeted sequences corresponded to a region of the DNA polymerase gene containing multiple conserved amino acid motifs. Herpesvirus genomic DNA fragments (222-244 bp) were amplified from 11 lesions by nested PCR and from eight lesions ( approximately 730 bp) using direct PCR from US cetaceans. Fragments of various sizes were also amplified from skin, spleen and blood of a German dolphin. Sequencing and BLAST analysis of these DNA fragments indicated that alpha- or gammaherpesviruses were present in the cetacean lesions. Alphaherpesviruses were associated with skin lesions of three Atlantic bottlenose dolphins (Tursiops truncatus), while gammaherpesviruses were present in genital lesions of five Atlantic bottlenose dolphins, one Risso's dolphin (Grampus griseus), one dwarf sperm whale (Kogia sima) and one Blainville's beaked whale (Mesoplodon densirostris), as well as in one oral lesion from an Atlantic bottlenose dolphin. Phylogenetic analysis of deduced amino acid sequences showed that the cetacean alphaherpesviruses were most closely related to human alphaherpesviruses, namely, herpes simplex-1 and -2. On the other hand, cetacean gammaherpesviruses were most closely related to Rhadinoviruses. These novel cetacean herpesviruses appeared to be distinct from known herpesviruses of marine and terrestrial vertebrates. The sequencing data strongly suggest that these viruses are most likely cetacean specific and possibly have coevolved with their cetacean hosts.