New polyacetylenic alcohols with a C(45) carbon skeleton (2) and with an enone moiety in the alkyl chain (C(46), 1) were isolated from the marine sponge Petrosia sp. The gross structures of 1 and 2 were established by spectral methods, and the absolute stereochemistry was determined by the modified Mosher's method. Compounds 1 and 2 displayed considerable cytotoxicity against a small panel of human solid tumor cell lines. Significant inhibitions on DNA replication by 1 and 2 were also observed which could be explanative of their cytotoxicity.