Certain functional analogs of amino acids were examined for their capacity to inhibit chemically induced expression of endogenous xenotropic retrovirus from Kirsten sarcoma virus transformed BALB/c (K-BALB) mouse cells. Partially synchronized cells cultured with aminoethylcysteine (AEC), parafluorophenylalanine (PFA), or valinol, and subsequently induced with either 5-iododeoxyuridine (IUdR), cycloheximide, or histidinol, showed inhibition of virus activation. Inhibition was concentration- and time-dependent (1 to 4 h) and not a consequence of cytotoxicity. Inhibition was competed out by the analogous amino acid and was specific to the induction process. After a 4 h analog treatment, heteronuclear RNA synthesis was reduced 24, 38, and 35% by PFA, AEC, and valinol, respectively, whereas cycloheximide or actinomycin D reduced synthesis by 60 and 90%, respectively; therefore, the analogs did not seem to inhibit induction through a general transcriptional block. Culture of cells in the presence of the analogs resulted in an abrupt reduction (70 to 90%) in DNA synthesis. Using synchronized cells, it was found that 0.1 mM AEC added in G1 phase and followed by IUdR induction almost totally inhibited virus expression. No inhibition was observed when AEC was added during S phase concomitantly with the inducer. AEC added to synchronous cells in G1 phase inhibited the progression of cells into S phase and the onset of DNA synthesis. The results show that K-BALB cells have an AEC-sensitive restriction point in G1 phase that might relate to the effects amino acid analogs have on cell replication and S phase dependent gene expression, as well as subsequent differentiation.