<b><i>Objective:</i></b> Interferon (IFN)β treatment is a mainstay of relapsing-remitting multiple sclerosis (RRMS) immunotherapy. Its efficacy is supposedly a consequence of impaired trafficking of inflammatory cells into the central nervous system and modification of the proinflammatory/antiinflammatory cytokine balance. However, the effects of long-term monotherapy using various IFNβ preparations on cytokine profiles and the relevance of these effects for the therapy outcome have not yet been elucidated. <b><i>Methods:</i></b> Changes were compared in serum levels of TNFα, IFNγ, interleukin (IL)-6, IL-10 and nitrite between RRMS patients given 3-year treatment with intramuscular IFNβ-1a (30 μg once a week) or subcutaneous IFNβ-1b (250 μg every other day). Only the data from patients who completed the 3-year study (n = 20 and n = 18, respectively) were analyzed. <b><i>Results:</i></b> Three-year IFNβ-1a or IFNβ-1b monotherapy reduced serum nitrite levels by 77 and 71%, respectively, lowered multiple sclerosis relapse annual rate by 70 and 71%, respectively, and significantly and similarly lowered Expanded Disability Status Scale scores in both study groups (by 0.9 on average). The two monotherapies showed little if any effect on cytokine levels and cytokine level ratios after the first year, but exerted diverging effects on these indices later on; the only exception was the IFNγ/IL-6 ratio that showed a monotonous rise in both study groups over the entire study period. <b><i>Conclusion:</i></b> During long-term IFNβ monotherapy, the levels of the studied cytokines show no relevance to the course of RRMS and neurological status of patients, whereas there seems to be a link between these clinical indices and the activity of nitric oxide-mediated pathways.