An association between Epstein-Barr virus (EBV) infection and systemic lupus erythematosus (SLE) has been suggested from previous serologic evidence. Since most adults in Taiwan are EBV-infected, seroepidemiologic studies based on standard assays for EBV are unlikely to dissociate SLE patients and control groups. We reexamine this question by using novel methodologies in which IgA anti-EBV-coded nuclear antigens-1 (EBNA-1) and IgG anti-EBV DNase antibodies were analysed by ELISA, and EBV viral loads were detected by real-time quantitative PCR for 93 adult SLE patients and 370 age-, sex- and living place-matched healthy controls in Taiwan. The specificities of antibodies for extractible nuclear antigens were determined by Western blot. Our results show that IgA anti-EBV EBNA1 antibodies were detectable in 31.2% SLE patients but only in 4.1% of controls (odds ratio [OR] = 10.72, 95% confidence interval [CI] = 5.19-22.35; P < 10(-7)), IgG anti-EBV DNase antibodies were detected in 53.8% SLE patients but only in 12.2% controls (OR = 8.40, 95% CI = 4.87-14.51; P < 10(-7)). EBV DNA was amplifiable from the sera of 41.9% SLE patients but from only 3.24% controls (P < 0.05). A significant association of IgG anti-EBV DNase antibodies with anti-Sm/RNP antibodies was observed (P < 0.005). The higher seroreactivity and higher copy numbers of EBV genome indicated association of EBV infection with SLE in Taiwan.