We have previously shown that topoisomerase I (topo I) antagonist inhibited retrovirus replication. Since tyrphostins, synthetic compounds and protein tyrosine kinases (PTKs) blockers, inhibited topo I activity (manuscript in preparation) we examined their ability to inhibit Moloney murine leukemia virus (Mo-MuLV) replication. We found that non-cytotoxic doses of tyrphostin derivatives (AG-555, AG-18) blocked or substantially reduced Mo-MuLV replication in acute or chronically infected NIH/3T3 cells. Our experiments suggest that the antiviral effect of these tyrphostin derivatives was not the result of antiproliferative activity. However, the tyrphostin derivatives used in our present investigation differ in their ability to inhibit Mo-MuLV replication. Furthermore, as expected from stereospecific competitive inhibitors, the antiviral effect is not a general characteristic of all tyrphostin derivatives, since AG-213 does not affect Mo-MuLV replication. Our results indicate that these tyrphostin derivatives may represent a novel class of antiretroviral drugs.