Summary An optimal procedure to synchronize the life cycles of the individuals in a population of human cells, cultured in vitro, by repeated temporary inhibition of the DNA synthesis was elaborated. Cultures in the logarithmic growth phase were treated three times with 7.5 m M thymidine dissolved in medium. Each treatment was terminated after 24 hr by washing the cultures with control medium. Between two successive treatments the cells were kept in the control medium for 14 hr. The peak of the mitotic index in the synchronized culture was found to be about 12 times as high as the index in controls. The necessity of more than one treatment was demonstrated. Some problems concerning multiple treatment are discussed. A cloning experiment showed that only little irreversible damage is caused by the synchronizing treatment; microscopically no cellular abnormalities were observed.