Using fluorophenylalanine (FPA) to interfere with functional viral protein synthesis, we have investigated the complex transcriptional and post-transcriptional controls that operate in cells infected with frog virus 3. Our previous data, obtained by polyacrylamide gel electrophoresis of viral RNAs and proteins, showed that the addition of FPA at the beginning of infection completely prevented the synthesis of late viral RNAs and late viral proteins and blocked the normal progressive decline in the rates of synthesis of two quantitatively different classes (class I and class II) of early proteins. These results indicated that the initiation of late RNA and late protein syntheses, as well as the post-transcriptional regulation of early protein synthesis, was under the control of virus-specific proteins (D. B. Willis, R. Goorha, M. Miles, and A. Granoff, J. Virol. 24:326-342, 1977). In this communication, we show that the viral protein required to "turn on" the synthesis of late RNAs and late (class III) proteins was made within 1 to 1.5 h postinfection (p.i.); when we added FPA after this time, we observed the synthesis of all of the late macromolecules. The data also suggest that another viral protein, separate from the "turn-on" protein, controlled the abundance of late RNAs. In addition, at least two separate proteins were involved in the post-transcriptional regulation of two classes of early proteins. When FPA addition was delayed until 2 h p.i., the rate of synthesis of class I proteins (which normally peaked at 2 h p.i.) was reduced by 6 h p.i. just as in a normal infection, but the rate of synthesis of class II proteins (which normally reached a maximum at 4 h p.i. before declining) was reduced only when we waited until 3 or 4 h p.i. to add FPA. These experiments corroborate and extend previous evidence for the existence of numerous viral regulatory proteins in the control of frog virus 3 gene expression at the transcriptional and post-transcriptional levels.