A phi 29 DNA fragment containing gene 2, coding for a phi 29-specific DNA polymerase required for the formation of the terminal protein p3-dAMP initiation complex, the first step in phi 29 DNA replication, has been cloned in plasmid pPLc28 under the control of the pL promoter of bacteriophage lambda. Four polypeptides of Mr 68 000, 5800 and 3400 and less than 2000 were labelled with [35S]methionine after heat induction. The protein of Mr 68 000 had the size expected for protein p2 and it accounted for about 2% of the de novo synthesized protein. Protein p2 synthesized in Escherichia coli was shown to be stable and biologically active. Its enzymatic activity could be assayed by the in vitro formation of the protein p3-dAMP initiation complex when complemented with extracts from Bacillus subtilis infected with a phi 29sus2 mutant or with extracts from E. coli harbouring gene 3-containing recombinant plasmids. Moreover, protein p2-containing E. coli extracts could catalyze the initiation reaction in vitro when complemented with highly purified protein p3.