Various attempts have been made to improve Epstein-Barr virus (EBV) serodiagnosis by developing more practical and objective methods than immunofluorescence-based assays. In the present study, the performance of immunoblot-based assays were evaluated by comparing the results obtained by the gold standard immunofluorescence antibody (IFA) test for the detection of IgM and IgG antibodies against EBV viral capsid antigen (anti-VCA). Serum samples of 277 patients admitted to Ege University Hospital for routine EBV diagnosis were included in the study. The age range of the patients was 3 months-89 years (mean 28 years) and 104 of them were females and 173 were males. All the samples were assayed by commercial immunoblot (Euroline IgM and IgG; Euroimmun, Germany) and IFA (EBV-CA IgG and IgM, Euroimmun, Germany) methods. Crosstabulation, chi-square test and phi (phi) measures in SPSS 16.0 statistical package programme were used for data analysis. Of the 216 samples that were interpreted as positive with immunoblot-based IgM assay, only 34 (15.7%) were confirmed as positive with IFA, whereas 162 (75%) were negative, and 20 (9.3%) were equivocal (phi = 0.167; low correlation). Of the 85 samples that were anti-VCA IgG positive with immunoblot assay, 82 (96.5%) were positive, 2 (2.3%) were negative and 1 (1.2%) were equivocal with IFA (phi = 0.441; significant correlation). When the indeterminate results obtained by IFA test were excluded from the evaluation, the correlation between immunoblot VCA IgG and IFA IgG was 85.4% (88/103) and between immunoblot VCA IgM and IFA IgM was 27.3% (69/253). When the intensities of bands were evaluated for IgM testing, it was noted that as the intensity of the bands increased (1+ to 3+), IFA VCA IgM reactivity rates increased (from 9.9% to 29.5% for p19 band; from 24% to 85.7% for gp125 band). For immunoblot VCA IgM testing, 165 samples were found to be positive only for VCA p19 band. Of these samples, 135 (81.8%) were negative, 15 (9.1%) were positive and 15 (9.1%) were equivocal with IFA. It is observed that even though immunoblot assays with automated blotting and scanning systems can be a convenient alternative to immunofluorescence assay, the rate of false positivity obtained for VCA IgM was high (75%). It was concluded that in laboratories which apply immunoblotting as a primary screening test for EBV serodiagnosis, the positive VCA IgM results (particularly isolated p19 band positivity) and the presence of low intensity bands, should be confirmed by IFA testing.