Human cytomegalovirus (CMV) is a part of Herpesviridae family. In subjects with a normal immune response, the primary infection is asymptomatic or causes a disease, similar to mononucleosis. With immunocompromised individuals, due to the latent nature and the prevalence of the virus, CMV presents a high risk for severe infection or even death of the patient. CMV infection poses a major risk in patients with solid-organ transplantation after the end of antiviral prophylaxis. With in vitro determination of the T cell immune response to CMV we can assess the risk of late onset of CMV infection and adapt the treatment individually so that we can avoid subsequent complications. The purpose of our study was to introduce an intracellular method for determining interferon gamma (IFN-γ) production in T lymphocytes when stimulated with various CMV antigens. The study included 8 healthy volunteers who had already come into contact with the virus. The levels of IgG and IgM antibodies against CMV were determined in the blood of patients and healthy subjects and the production of IFN-γ after stimulation with different antigens was measured on a flow cytometer. In our study, we tested various of CMV peptides that were stimulated by T lymphocytes and found that US3 is the best stimulating peptide for CD4 + and CD8 + population of T lymphocytes. Measurement of a specific T cell response to CMV by measuring intracellular IFN-γ in T lymphocytes is important in deciding treatment of patients especially when lymphocyte concentration is decreased and IFN-γ cannot be determined by the commercial QuantiFERON-CMV assay.