Xenopus egg extract was fractionated by gel-filtration column chromatography and DNA synthetic activity was examined using double-stranded DNA as a template. The major activity eluted had an apparent molecular mass of about 300 kDa. Product analyses showed that de novo DNA synthesis occurs with formation of replication bubbles, thereby suggesting that this fraction catalyzes the initiation of DNA replication. Activities of DNA polymerase α-primase and DNA helicase overlapped with the DNA synthetic activity, but the elution profiles of the enzymes differed from that of the DNA synthetic activity. Therefore, this 300-kDa fraction may contain a component which differs from the above enzymes yet is essential for initiation of DNA replication.