The RegA protein of bacteriophage T4 is a translational repressor that regulates expression of several phage early mRNAs. We have cloned wild-type and mutant alleles of the T4 regA gene under control of the heat-inducible, plasmid-borne leftward promoter (PL) of phage lambda. Expression of the cloned regA+ gene resulted in the synthesis of a protein that closely resembled phage-encoded RegA protein in biological properties. It repressed its own synthesis (autogenous translational control) as well as the synthesis of specific T4-encoded proteins that are known from other studies to be under RegA-mediated translational control. Cloned mutant alleles of regA exhibited derepressed synthesis of the mutant regA gene products and were ineffective in trans against RegA-sensitive mRNA targets. The effects of plasmid-encoded RegA proteins were also demonstrated in experiments using two compatible plasmids in uninfected Escherichia coli. The two-plasmid assays confirm the sensitivities of several cloned T4 genes to RegA-mediated translational repression and are well-suited for genetic analysis of RegA target sites. Repression specificity in this system was demonstrated by using wild-type and operator-constitutive translational initiation sites of T4 rIIB fused to lacZ. The results show that no additional T4 products are required for RegA-mediated translational repression. Additional evidence is provided for the proposal that uridine-rich mRNA sequences are preferred targets for the repressor. Surprisingly, plasmid-generated RegA protein represses the synthesis of some E. coli proteins and appears to enhance selectively the synthesis of others. The RegA protein may have multiple functions, and its binding sites are not restricted to phage mRNAs.