Acute infection of cattle with bovine herpesvirus 1 (BHV-1) represses cell-mediated immunity, which can lead to secondary bacterial infections. Since BHV-1 can induce apoptosis of cultured lymphocytes, we hypothesized that these virus-host interactions occur in cattle. To test this hypothesis, we analyzed lymph nodes and peripheral blood mononuclear cells (PBMC) after calves were infected with BHV-1. In situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining of lymphoid tissues (pharyngeal tonsil, cervical, retropharyngeal, and inguinal) was used to detect apoptotic cells. Calves infected with BHV-1 for 7 days revealed increased apoptotic cells near the corticomedullary junction in lymphoid follicles and in the subcapsular region. Increased frequency of apoptotic cells was also observed in the mucosa-associated lymphoid tissue lining the trachea and turbinate. Immunohistochemistry of consecutive sections from pharyngeal tonsil revealed that CD2(+) T lymphocytes were positive for the BHV-1 envelope glycoprotein gD. The location of these CD2(+) T lymphocytes in the germinal center suggested that they were CD4(+) T cells. Electron microscopy and TUNEL also revealed apoptotic and herpesvirus-infected lymphocytes from this area. Fluorescence-activated cell sorting analyses demonstrated that CD4(+) and CD8(+) T cells decreased in lymph nodes and PBMC after infection. The decrease in CD4(+) T cells correlated with an increase in apoptosis. CD4(+) but not CD8(+) lymphocytes were infected by BHV-1 as judged by in situ hybridization and PCR, respectively. Immediate-early (bovine ICP0) and early (ribonucleotide reductase) transcripts were detected in PBMC and CD4(+) lymphocytes prepared from infected calves. In contrast, a late transcript (glycoprotein C) was not consistently detected suggesting productive infection was not efficient. Taken together, these results indicate that BHV-1 can infect CD4(+) T cells in cattle, leading to apoptosis and suppression of cell-mediated immunity.