The objective of this work was to compare the seroprevalence of cytomegalovirus in an unexposed and exposed population, both working in a hospital, and to study the occupational risk factors related to seropositivity, while taking personal risk factors into account. We conducted a cross-sectional study in a French hospital over a period of 12 months. The overall seroprevalence among the 550 subjects was 49.5%. The multivariate analysis showed that seropositivity was significantly associated with age (36-43 years: odds ratio [OR] = 1.7; 95% confidence interval [CI]: [1.1-2.8]) and working as a pediatric nurse's aide (OR = 1.8; 95% CI: [1.1-2.8]). This study confirms the need to improve prevention procedures in the workplace, including screening, information, and hygiene rules.