Introduction and objective Th17 lymphocytes and their cytokines, interleukin 17A (IL-17A), IL-17F and IL-22, participate in the response to extracellular bacteria and in the autoimmunity and may be engaged in the pathogenesis of Lyme borreliosis. Concentrations were measured of IL-17A, IL-17F and IL-22 in the supernatant of the peripheral blood mononuclear cells (PBMC) culture stimulated with Borrelia burgdorferi sensu lato ( B. burgdorferi ). Material and Methods The study group consisted of 13 patients with early disseminated and late Lyme borreliosis and a control group of 7 healthy persons. PBMC cultures were stimulated for 48 hours with B. burgdorferi spirochetes of three pathogenic species: B. burgdorferi sensu stricto, B. afzelii or B. garinii , in the multiplicity of infection 10:1. Concentrations of Th17 cytokines IL-17A, IL-17F and IL-22, as well as Th2/immunoregulatory cytokine IL-10 were measured with ELISA assays. Results Expression of IL-17A, IL-17F and IL-22 increased under stimulation, simultaneously with the increased IL-10 expression. Concentration of IL-17F tended to be lower in early neuroborreliosis than in late Lyme borreliosis and than in controls. B. afzelii elicited higher expression of IL-17A than the other two species. Conclusions IL-17A, IL-17F and IL-22 are synthesized simultaneously by PBMC stimulated with B. burgdorferi . There is no antagonism between Th17 response and IL-10 expression. The role of Th17 cytokines seems to differ depending on the clinical stage of Lyme borreliosis and on the B. burgdorferi species.