OOP RNA is a major short (77 bases) transcript that is made from bacteriophage lambda DNA both in vivo and in vitro. OOP RNA is synthesized in the opposite direction to mRNA for the lambda cII gene, and the final 55 bp of the OOP region overlaps the 3' end of the cII gene. We find that a multicopy plasmid containing an OOP DNA fragment inhibits cII expression from a derepressed prophage by approximately 100-fold, using an in vivo assay in which cII protein activates galactokinase synthesis from a cII-dependent promoter on a multicopy plasmid. A large inhibitory effect is also observed when the po promoter for OOP RNA is replaced by the strong lambda pL promoter, but not when po is deleted. Plasmids that provide a large excess of "anti-OOP" RNA (RNA that is complementary to OOP RNA) make OOP RNA a less effective inhibitor of cII expression. Inhibition by the OOP DNA plasmid is not observed in an Escherichia coli strain deficient in RNase III. We propose that the 3' end of cII mRNA and OOP RNA form a double-stranded complex that is a substrate for the host enzyme RNase III, resulting in degradation of cII mRNA. Deletion studies on the OOP DNA plasmid indicate that no specific sequence between the promoter and terminator stem structure is required for the inhibitory effect. Lambda cII expression from an induced prophage is increased twofold in the presence of a large excess of anti-OOP RNA. This experiment, in which the prophage is the sole source of OOP RNA, suggests a physiological role for OOP RNA in regulating cII-gene expression.