Epidemiology of cytomegalovirus (CMV) infection varies widely, depending on ethnicity and socioeconomic status. A seroepidemiological survey was conducted to determine CMV infection among pregnant women in Nagasaki Prefecture, Japan. We measured serum CMV-specific immunoglobulin (Ig) M and IgG at the first and third trimesters. IgG avidity was determined when both CMV-IgG and CMV-IgM were positive. Of 339 pregnant women, 296 (87.3%) were CMV-IgG-positive at the first trimester. Of 267 paired sera, one (0.37%) had CMV-IgG seroconversion, another one (0.37%) had CMV-IgM seroconversion, and 12 had both CMV-IgG and CMV-IgM, two (0.75%) of whom had low IgG avidity, suggesting recent infection. Thus, the incidence of primary CMV infection during pregnancy was 0.74-1.5%. Assuming the rate of in utero transmission following maternal primary infection to be approximately 40%, the incidence of congenital infection is estimated to be 0.3-0.6%. Although CMV seroprevalence among pregnant women has been decreasing in industrialized regions including other parts of Japan, CMV-seroprevalence remains high in Nagasaki. Thus, epidemiology of CMV infection seems variable within Japan, a country generally considered to be ethnically and socioeconomically homogeneous. However, 40-80 infants may be congenitally infected and 15-27% (or 6-22) of them may ultimately suffer from certain neurological sequelae annually in Nagasaki Prefecture, so where annual live births are approximately 13,300, congenital CMV infection seems to be a significant public health problem in such an apparently low-risk region as Nagasaki.