Using an in vivo complementation system, we conducted a mutational analysis of the bacteriophage Q beta readthrough cistron. In the Q beta cDNA-containing plasmid, pQ beta m100, we constructed six defined Q beta deletion cDNA genomes, each missing between 86 and 447 nucleotides from within the readthrough cistron. These deletion plasmids were introduced into host cells that are constitutively supplied with Q beta readthrough protein from the plasmid pQ beta RT. Under these conditions, all six deletion genomes spontaneously generated phage particles, each exhibiting a characteristic plaque phenotype and virus forming potential. Isolated readthrough-defective phage particles were subsequently used to infect host cells that carried helper readthrough protein. Passaged viruses yielded both larger plaques and higher titers, compared with those of the parent phages. Sequence analysis revealed that the genomes of the passaged viruses had deleted additional regions of readthrough RNA sequence. We discuss the possibilities that (1) the disruption of a well-defined structural domain in Q beta RNA was selectively disadvantageous to phage infection, and that (2) the evolved viral populations were selected by virtue of their ability to restore critical integrity of short and/or long-range nucleotide interactions within this region of Q beta RNA.