An unusual peptide-encoding sequence, called H10, and several derivatives of this sequence were previously isolated from a random peptide library screened by phage display during drug discovery protocols. The H10 family of sequences had the unusual property of being expressed despite the absence of an open reading frame. When these sequences were fused to a reporter lacZ gene in all three frames, beta-galactosidase was expressed not only from the parental non-open reading frame, consistent with the original isolations, but also from the frame -1 to the parental. This unexpected translation in a second reading frame could result from either a recoding event or from an internal translation initiation event. In order to elucidate which type of event, a genetic approach was selected to eliminate a potential downstream initiator site within the H10 sequence. This report provides strong evidence that translation in the -1 frame in this family of sequences is indeed originating from a downstream translation initiation event. Unexpectedly, the mutation eliminating the downstream initiation event in the -1 frame simultaneously elevated expression in the original non-open reading frame.