Abstract Two-stranded calf thymus DNA is first incubated with highspeed supernatant fraction prepared from regenerating rat liver with and without EDTA and then reisolated. None of these two DNA's are found to be active as a primer without heating in the system involving the use of purified calf thymus polymerase. They are however as active as calf thymus DNA in the other system where polymerase present in the regenerating rat liver supernatant fraction is used. On the basis of the above observations it is concluded that the polynucleotide chains of DNA, held together by hydrogen bonds between the bases (AT, GC) are never completely separated as single-strand DNA molecules before DNA synthesis actually starts. It is rather assumed that DNA synthesis starts at the two ends of the double-helical structure of primer DNA and the process is continued in which successive breakdown of hydrogen bonds between the pair of bases accompanied by the formation of new hydrogen bonds between the unpaired bases and incoming bases, satisfying the AT, GC rule has been supposed to occur until two new molecules of two-stranded DNA are formed from one primer DNA molecule.