The nonstructural protein NS-1 of minute virus of mice (MVMp), an autonomous parvovirus, trans-inhibits the replication of a chimeric plasmid containing the SV40 origin of replication (ori) embedded in the MVMp genome. It appears that a 157-bp 5' proximal sequence of MVMp DNA is sufficient, in the presence of NS-1, to cause the inhibition of DNA replication driven by the SV40 ori placed on the same molecule. This effect is not dependent on the orientation of the MVMp target sequence and results from both a reduced level of utilization of SV40 ori and the blockage of progressing replication forks at the level of the target. Furthermore, replication driven by Epstein-Barr virus origin (oriP) is trans-inhibited by MVMp but this inhibition does not require the presence of parvoviral sequences in cis. On the basis of sequence homologies between EBV oriP and MVMp 5' terminal sequence, it is proposed that the direct or indirect interaction of NS-1 with parvovirus-like sequences present in heterologous viral and possibly also cellular genomes may result in an inhibition of DNA replication.