Human papillomavirus (HPV) virus-like particle (VLP) vaccines are highly effective at preventing viral infections and the development of precancerous lesions through the induction of high-titer neutralizing antibodies and strong cell-mediated immune responses. Women taking combined oral contraceptives (COCs), however, show large variabilities in the magnitudes of their antibody responses. The goal of the present study was to determine the effects of 17beta-estradiol (E2) and progesterone (P4) alone and in combination on the cellular immune response to HPV type 16 (HPV-16) VLPs in vitro. Peripheral blood mononuclear cells (PBMCs) from healthy donor women were stimulated in vitro with HPV-16 VLPs (2.5 microg/ml) in the presence of E2 and P4 administered either alone or in combination; and lymphoproliferation, cytokine production, transcription factor expression, and steroid hormone receptor expression were analyzed. HPV-16 VLPs significantly increased the levels of lymphoproliferation, proinflammatory cytokine (gamma interferon [IFN-gamma], interleukin-1beta [IL-1beta], IL-2, IL-6, IL-8, IL-12p70, IL-17, tumor necrosis factor alpha [TNF-alpha]) production, anti-inflammatory cytokine (IL-1ra, IL-10) production, and the expression of Eralpha and Erbeta but decreased the levels of Foxp3 expression and production of transforming growth factor beta (TGF-beta). Exposure of PBMCs to E2 and P4 either alone or in combination significantly decreased the levels of lymphoproliferation and production of proinflammatory cytokines (IFN-gamma, IL-12p70, TNF-alpha) but increased the levels of production of IL-10 and TGF-beta and the expression of Foxp3 in response to HPV-16 VLPs. Treatment of cells with biologically relevant concentrations of sex steroid hormones suppressed the inflammatory response and enhanced the regulatory response to HPV-16 VLPs, which may have implications for predicting the long-term efficacy of HPV vaccines, adverse events, and cross-protection among women taking COCs.