Replication rate and replicon sizes in chromosomal DNA of in vitro cultured diploid D. melanogaster cells were determined using autoradiography of 3H-thymidine labeled DNA. Synthesis of DNA in euchromatic and heterochromatic regions of Drosophila diploid cells occurs at different periods of the S phase which last 10 h. During the first 4 h the synthesis is observed only in euchromatic regions. The heterochromatic synthesis starts shortly before the synthesis in euchromatic regions is completed and lasts for 6 h until the end of the S phase. The cells were synchronized by 5-fluorodeoxyuridine which blocked the diploid cell DNA synthesis. Synthesis was found to start simultaneously in most euchromatic replicons. In the majority of the replicons the synthesis started at a single point and proceeded bidirectionally. The average rate of DNA synthesis per fork was 12.5 mum/h (38kb). The mean distance between the middle points of adjacent labeled regions was 70 mgm (210 kb). The size of most replicons ranged from 40 to 120 mum. - These estimates do not apply to the heterochromatic portions of the D. melanogaster genome since the measurements have been carried out on DNA preparations obtained during the first 2 h of the S phase. - On the average, a replicon can consist of 7 chromomeres since the size of a replicon in diploid cell chromosomal DNA and DNA length of a polytene chromomere average 210 and 30 kb, respectively.