T cells simultaneously producing multiple cytokines and possessing cytotoxic capacity termed polyfunctional cells (PFCs) are increasingly recognized as the immune correlate of protection against pathogenic viruses. We investigated co-expression of four cytokines (interferon-γ, macrophage inflammatory protein 1-α, tumour necrosis factor-α and interleukin-2) and degranulation capacity (CD107a surface expression) of Epstein–Barr virus (EBV) -specific CD4+ and CD8+ T cells upon stimulation by overlapping peptides of EBV lytic (BZLF1) and latent (EBNA1, EBNA3 and LMP2) proteins, in 20 healthy Chinese long-term carriers. Two patients with post-transplant lymphoproliferative disorder (PTLD), who had impaired T-cell immunity, were studied for comparison. Both EBV-specific CD4+ and CD8+ PFCs were readily generated in long-term carriers and showed immunodominance hierarchies of latent proteins (EBNA1 > EBNA3/LMP2 and EBNA3 > LMP2 > EBNA1 for CD4+ and CD8+ T cells, respectively), as evidenced by a higher proportion of PFCs generated by immunodominant EBV proteins than by subdominant viral proteins. In contrast, the proportion of EBV-specific PFCs was markedly decreased in patients with PTLD. The EBV-specific PFCs produced more cytokine per cell than single-functional T cells and comprised different subsets. Five-functional CD4+ and CD8+ T cells were detected and four-functional CD4+ T cells were mainly CD107a negative and expressed all four cytokines whereas four-functional CD8+ T cells were mainly CD107a positive and expressed three of the four cytokines (interleukin-2-negative). We conclude that EBV-specific PFCs are generated in much higher proportions in the long-term carriers than in the patients with PTLD and maintain the immunodominant characteristics of the virus.