The gene 17 of the Bacillus subtilis phage phi29 is known to be involved in the viral DNA replication in vivo. In this paper, we show that the presence of protein p17 is required when phage infection occurs at a low multiplicity of infection (moi), which is probably the natural condition for infection, but is dispensable at a high moi. Gene 17 has been cloned in an Escherichia coli expression vector and protein p17 purified. A stimulatory effect of protein p17 was demonstrated under in vitro conditions required to amplify phi29 DNA, starting with a low amount of input DNA. We propose that p17, which is synthesized early after infection, is required at the very beginning of the phage amplification, conditions in which a low number of viral DNA molecules enter the host cell, possibly to recruit the limiting amount of initiation factors at the replication origins. Once the infection process is established and the other replication proteins reach optimal concentration, p17 becomes dispensable.