The aims of the study were to evaluate the occupational risk of cytomegalovirus (CMV) infection in kindergarten teachers working with children aged 2.5-6 years, and to determine occupational risk factors within the occupation of kindergarten teaching. A cross-sectional seroprevalence study was conducted in 211 kindergarten teachers and 283 administrative workers. Relevant confounding factors were considered. Overall seropositivity rates ranged from 16.4% in childless women to 33.7% in women with one child or more. Raising own children was the major risk factor for CMV seropositivity: adjusted OR 2.25. Kindergarten teaching showed to have a significantly increased CMV seropositivity rate: adjusted OR 1.54. Among kindergarten teachers, washing hands at school, number and age of the pupils, and seniority had no significant influence on seropositivity. The results indicated an increased risk of CMV infection in kindergarten teachers and an insufficiency of hygienic measures to prevent seropositivity.