The role of different cytokines and cells of immune system in the pathogenesis of chronic GVHD (cGVHD) is still controversial. Earlier studies, which were either retrospective or analysed one or a few factors, did not show unequivocal results. We prospectively evaluated cytokine levels and lymphocyte subsets in 30 patients who underwent Allo-SCT to investigate their possible correlation with cGVHD. Levels of IL-4, IL-6, IL-10, IFN-gamma, tumour necrosis factor-alpha (TNF-alpha) and its soluble receptors were assessed by ELISA in 30 patients at different times after SCT. Lymphocyte subsets were evaluated by flow cytometry in peripheral blood at the same times as cytokines. A multivariate analysis was performed using principal component analysis and multi-factor ANOVA (analysis of variance). Eighteen patients developed cGVHD at a median time of 6 months (range, 5-9) after SCT. In multivariate analysis, we observed a correlation between cGVHD and clusters of cytokines and lymphocyte subsets from the third to the sixth month after SCT. These clusters changed their composition over time, but they constantly included natural killer (NK) and CD152+ T cells as negative predictors of cGVHD. TNF-alpha prevailed among other cytokines before the onset of cGVHD. This prevalence could be related partly to the defect of immunoregulatory cells.