The enveloped bacteriophage phi6 has been shown to be an interesting model system for the study of chemical agents that might have specific antiviral effects against lipid-containing mammalian viruses. In this report, we describe two types of antiviral activity exhibited by several fatty acids against bacteriophage phi6. Oleic acid (18:1) and palmitoleic acid (16:1) were potent inactivators of the virus. Treatment with either fatty acid at 50 mug/ml at 25 or 0 degrees C for 30 min reduced the virus titer to about 0.1% of the initial titer. Oleic acid at a concentration as low as 3 mug/ml ( approximately 10(-2) mM) reduced the virus titer to <1% of the initial titer within 30 min. Ultracentrifugation analyses of (14)C-amino acid- and (32)P-labeled virus treated with oleic acid indicated that the virion is largely disassembled by the treatment. Myristic acid (14:0) and palmitic acid (16:0) did not inactivate phi6 at 50 mug/ml, but nevertheless did prevent phi6 plaque production. Single-step virus growth experiments in which fatty acid was added at various times before or after infection indicated that it was an early stage of the phi6 replication cycle that was inhibited by the presence of myristic acid and that the inhibition occurred only if the myristic acid concentration in the extracellular growth medium was greater, similar10 mug/ml. phi6 could attach to its host cell in the presence of myristic acid at 50 mug/ml. We conclude that the fatty acids that prevent phi6 replication probably do so by interfering with the entry of the viral genome into the host cell.