Cerulenin, an inhibitor of de novo fatty acid (and cholesterol) biosynthesis, has been shown to significantly decrease (greater than 75%) the amount of Moloney murine leukemia virus (MMuLV) released into the culture medium of chronically infected mouse fibroblasts (I. Katoh, Y. Yoshinaka, and R.B. Luftig, 1986, Virus Res., in press). In order to clarify the mechanism by which this decrease in virus production occurs, we analyzed the kinetics of gag and env coded protein synthesis in M-MuLV infected, cerulenin-treated cells by immunoprecipitation with monospecific antisera to p30, p12, p10, gp70, and p15(E). We found that in pulse (15 min-2 hr)-chase (0-4 hr) experiments the cleavage of not only Pr65gag to p30 and other gag coded proteins but Pr80env to gp70 and Pr15(E) as well, was greatly reduced by cerulenin treatment. Further, since the total amount of label in the Pr65gag and Pr80env bands remained about the same or was slightly decreased in 2-hr pulsed, cerulenin-treated cells, this suggests that cerulenin decreases virus production, in part, by inhibiting the cleavage of both precursor gag and env coded polyproteins during virus assembly and budding at the cell membrane. We also observed that at longer chase periods (4 hr), the effect of cerulenin could be partially overriden in that minor amounts of cleaved gag and env coded polyproteins were produced and assembled into virion particles. However, these particles contained abnormally large amounts of the uncleaved precursor Pr65gag, suggesting that maturation was incomplete. The above results suggest two independent, but not exclusive, possible mechanisms of cerulenin action to block M-MuLV production, viz. cerulenin decreases the pool of fatty acids, thereby inhibiting fatty acid acylation of Pr65gag, as well as Pr80env, and thus preventing the interaction between gag (the p15 antigenic determinant on Pr65gag) and env [the p15(E) antigenic determinant of Pr15(E)] coded gene products at the cell membrane needed for efficient virus assembly (M. Satake and R. B. Luftig, 1983, Virology 124, 259-273), and cerulenin inhibits one or more proteolytic enzymes responsible for the cleavage of Pr65gag and Pr80env.