CD4(+) T lymphocytes of patients with chronic kidney disease (CKD) are characterized by reduced levels of crucial surface antigens and changes in the cell cycle parameters. Recombinant human erythropoietin (rhEPO) normalizes their altered phenotype and proliferative capacity. Mechanisms leading to the deficient responses of T lymphocytes are still not clear but it is postulated that immunological changes are deepened by hemodialysis (HD). Study of activation parameters of CD4(+) T lymphocytes in hemodialyzed and predialysis CKD patients could bring insight into this problem. Two groups of patients, treated conservatively (predialysis, PD) and hemodialyzed (HD), as well as healthy controls, were included into the study; neither had received rhEPO. Proportions of main CD4(+)CD28(+), CD4(+)CD25(+), CD4(+)CD69(+), CD4(+)CD95(+), and CD4(+)HLA-DR(+) lymphocyte subpopulations and proliferation kinetic parameters were measured with flow cytometry, both ex vivo and in vitro. No differences were seen in the proportions of main CD4(+) lymphocyte subpopulations (CD4(+)CD28(+), CD4(+)CD25(+), CD4(+)HLA-DR(+), CD4(+)CD69(+), CD4(+)CD95(+)) between all examined groups ex vivo. CD4(+) T lymphocytes of HD patients exhibited significantly decreased expression of co-stimulatory molecule CD28 and activation markers CD25 and CD69 after stimulation in vitro when compared with PD patients and healthy controls. HD patients showed also decreased percentage of CD4(+)CD28(+) lymphocytes proliferating in vitro; these cells presented decreased numbers of finished divisions after 72 h of stimulation in vitro and had longer G0→G1 time when compared to healthy controls. CD4(+) T lymphocytes of PD patients and healthy controls were characterized by similar cell cycle parameters. Our study shows that repeated hemodialysis procedure influences phenotype and proliferation parameters of CD4(+) T lymphocytes.