A seroprevalence survey on measles, mumps, rubella and varicella was conducted on healthcare workers (HCWs) at Shimane University Hospital, Japan utilizing an enzyme immunoassay. Of 1811 HCWs tested, 91.8% were seropositive to measles, 92.1% to mumps, 89.5% to rubella and 96.3% to varicella. Sex-related differences in seroprevalence were found in rubella (males vs. females: 84.7 vs. 92.2%, P < 0.001). Moreover, males aged 30-39 years were most susceptible to rubella (22.4%), which may be attributed to the design of childhood immunization programmes in Japan. Individuals aged ≤ 29 years were more susceptible to measles (14.3%) and mumps (10.9%), compared to other age groups. There were no significant sex- and age-related differences in varicella seroprevalence. The physician occupational group was more susceptible to rubella, but no significant occupational-related difference was observed in the other diseases. Susceptible subjects, with negative or equivocal serological results were given a vaccine which induced seroconversion in most vaccinees. Seroconversion occurred more frequently in the equivocal group than in the negative group. These findings provide a new insight for the seroprevalence survey of vaccine-preventable diseases in Japanese HCWs with special reference to vaccine efficacy.