Isoprinosine is a synthetic purine derivative with immunomodulatory and antiviral properties, which result from an apparent in vivo enhancement of host immune responses. To evaluate the serum levels of certain cytokines during and after isoprinosine treatment, we assigned 10 healthy volunteers to receive isoprinosine 1 g, 3 times daily, 5 consecutive days weekly. Both treatment and follow-up phase last 3 weeks. Interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-10, and tumor necrosis factor-alpha (TNF-alpha) were measured in serum using commercial ELISA kits at baseline, 7th, 10th, 14th, 21st, 28th, 35th, and 42nd day. We observed an increase in serum levels of all measured cytokines at 7th to 10th day. The levels of IL-2 had another raise at 42nd day after drop to initial values (P < 0.05; P < 0.001, respectively). Those of IL-10 held up enhanced from 7th to 28th day of measurement (P < 0.01). There was a nearly flat line of values of TNF-alpha after initial slight increase at 10th day. We found a moderate negative correlation between IFN-gamma and IL-2, IL-10, and TNF-alpha (Spearman's r: -0.63, -0.62, -0.63; P < 0.05, respectively). We have demonstrated the immunomodulating properties of isoprinosine in healthy adults. It suggests resumption of the research with up-to-date methods to elucidate the mechanisms of action of inosine pranobex and maybe the other inosine compounds in different clinical settings.