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Bovine alphaherpesvirus 1 (BHV1) is an agent associated with reproductive disease in cattle. Viral pathogenicity is related to disorders such as temporary infertility, embryonic death, and abortions in affected animals. Considering that natural infections in genital organs of males are understudied, this investigation evaluated the presence of BHV1 in both testicular and epididymal tissues obtained from naturally infected bulls by the evaluation of the presence of the BHV1 genome and antigens. Sixty samples of blood and genital organs of 60 bulls that were not vaccinated against BHV1 were assayed. Fragments from testes and head, body, and tail of epididymides were processed and analyzed by nested PCR and immunofluorescence with confocal laser scanning microscopy. Also, the BHV1 gB glycoprotein gene of 14 positive samples was partially sequenced. The percentage of BHV1 presence obtained by the immunolocalization assay corresponded to 95.9% of the testes, 100% of the epidydimal tissue in the head and tail portions, and 98% of the epididymal body. The nested PCR assay detected the viral nucleic acid in 59.2% of the testicular tissue and in 65.3, 75.5, and 83.7% of epididymis head, body, and tail samples, respectively. The partial sequences analyzed presented 100% of identity with other BHV1 strains. Accordingly, BHV1 detection in testes and epididymides of naturally infected bulls suggests that these organs may be sources of viral infection for semen., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)