The authors tested their hypothesis that Epstein-Barr virus (EBV) infection is a risk factor for aqueous tear deficiency (ATD) by evaluating 38 ATD patients and 17 controls for serologic evidence of EBV infection. Aqueous tear deficiency was graded clinically as mild or severe. A linear trend toward elevated EBV capsid (P less than 0.05) and early antigen (P less than 0.001) titers was noted from control to severe ATD patients. Rubella and cytomegalovirus antibody titers were poorly correlated with EBV titers, suggesting that the elevated EBV antibodies in ATD patients were not due to nonspecific polyclonal B-cell activation. Epstein-Barr virus antigens were detected in two of six lacrimal gland biopsies from severe ATD patients with Sjögren's syndrome, but in none of the control glands. Aqueous tear deficiency patients were evaluated for immunologic dysfunction associated with EBV infection. Linear trends of elevated serum IgG (P less than 0.05), autoantibody and immune complex positivity (P less than 0.05), and reduced natural killer cell cytotoxicity (P less than 0.05) were found from controls to severe ATD patients. Furthermore, reduced T-helper lymphocyte counts (P less than 0.06) and an increased percentage of HLA-DR+ CD8 lymphocytes (P less than 0.05) were observed in severe ATD patients compared with the mild and control groups. A multivariate analysis of the data showed a significant correlation between severe ATD and elevated EBV early antigen titers, Sjögren's syndrome, and an increased percentage of HLA-DR+ CD8 lymphocytes. The authors' findings suggest that EBV infection may be a risk factor for development of ATD in a subset of ATD patients with greater disease severity, Sjögren's syndrome, and immunologic dysfunction.