Measurements of mucosal immune parameters in the uterine cervix are potentially influenced by numerous factors, including infections, endogenous and exogenous hormones, semen, and nicotine and its metabolites in cervical mucus. The objective of this study was to examine correlates of immunoregulatory cytokine mRNA expression in cervical cytology samples in a cross-sectional design. Samples, collected at study entry by cervical cytology brush from 368 women aged 13–21 enrolled in a longitudinal study of the natural history of human papillomavirus (HPV) infection, were tested by quantitative RT-PCR for expression of IFN-γ, IL-4, IL-10, and IL-12. In a multivariate analysis, elevated levels of IFN-γ, IL-10, and IL-12 were significantly (p < 0.05) associated with several variables, including current C. trachomatis infection, recent intercourse, and current oral contraceptive pill use. Suppressed IL-4 and IL-10 levels were associated with cigarette smoking within the last 24 h. Time since last menstrual period did not affect any of the cytokines; in a substudy of weekly cytokine variability, however, IL-10 showed a non-significant trend toward higher levels around the time of menstruation.