Little is known about childcare agency staff vaccination requirements, parents' perceptions of these requirements, or vaccine uptake in these populations. Methods: A questionnaire was administered to St Louis parents and childcare agency staff in fall of 2014. The [chi square] tests compared staff's versus parents' uptake of hepatitis A, pertussis, and seasonal influenza vaccines. Multivariate logistic regression was used to examine individuals being fully immunized (ie, having received influenza, hepatitis A, and pertussis vaccines). Results: Overall, 351 parents and staff from 23 agencies participated (response rate, 32%). One-third of staff (34.4%, n=33) and parents (37.6%, n=96) were fully immunized. Parents and staff were equally likely to have received the influenza vaccine (48.8% and 47.3%, respectively), but more staff received the hepatitis A vaccine (85.3% vs 67.5%, [chi square]=11.0, P.001), and more parents received the pertussis vaccine (66.5% vs 45.8%, [chi square]=12.5, P.001). Determinants of being fully immunized included having previously received the influenza vaccine, being offered the vaccines, belief that vaccination is important, having immunization recommendation awareness, and not having vaccine misperceptions. Conclusions: Childcare agency staff vaccination can protect employees and children from disease, but their uptake of vaccines needs improvement. Future interventions should be aimed at increasing uptake to lower disease transmission in childcare settings. (author abstract)