In the course of our search for bioactive metabolites from the marine sponges collected from Korean water, we found that the polyacetylenes of marine sponge, genus Petrosia, deliver significant selective cytotoxicity against several human tumor cell lines. The effects of polyacetylene on DNA replication were examined using simian virus 40 DNA replication system in vitro. We found that polyacetylenes inhibited DNA replication, and predominantly inhibited the initiation stage of DNA replication. Polyacetylenes inhibited the DNA cleavage by topoisomerase I, and also significantly reduced polymerase alpha-primase activity. The ssDNA binding activity of replication protein A was little affected by polyacetylenes. We suggest that polyacetylenes might inhibit proteins required to establish replication forks during the initiation reaction, and their cytotoxicities might be related to the inhibitory effect they have on this fundamental cellular process.