Background: This study aims to examine the impact of women's characteristics (demographics, risk behaviour, and beliefs) on the uptake of cervical cancer screening, taking practice characteristics (demographic and organizational) into account. Methods: Routinely collected data of screening status were sampled from electronic medical records of 32 Dutch general practices. Additionally, a questionnaire was sent to a sample of 2224 listed women—1204 screened, 1020 unscreened. We used a step-by-step, logistic, multilevel approach to examine determinants of the screening uptake. Results: Analyses of data for 1392 women (968 screened and 424 unscreened) showed that women's beliefs about cervical screening and attendance are the best predictors of screening uptake, even when demographic and organizational aspects are taken into account. Women aged 40–50 years who felt high personal moral obligation, who had only one sexual partner ever, and who were invited and reminded by their own general practice had the greatest likelihood of screening uptake. A non-response study was performed; the non-responders to the questionnaire (mainly unscreened) thought they had less risk of cervical cancer, were less motivated, less often intended to get future screening, and were more convinced that cervical cancer cannot be cured. Conclusion: To improve the uptake rate, we should focus on the personal moral obligation of eligible women, beliefs about the risks of cervical cancer, and available cures. Invitations and reminders within general practices enhance the uptake rate.