The genomic RNA of human astrovirus serotype 1 (HAst-1) contains three open reading frames (ORFs), 1a, 1b, and 2. ORF 1b is located downstream of, and overlaps, 1a, and it has been suggested on the basis of sequence analysis that expression of ORF 1b is mediated through -1 ribosomal frameshifting. To examine this possibility, a cDNA fragment containing the 1a-1b overlap region was cloned within a reporter gene and placed under the control of the bacteriophage SP6 promoter in a recombinant plasmid. Synthetic transcripts derived from this plasmid, when translated in the rabbit reticulocyte lysate cell-free system, specified the synthesis of polypeptides whose size and antibody reactivity were consistent with an efficient -1 ribosomal frameshift event at the overlap region. The HAst-1 frameshift signal has two essential components, a heptanucleotide slippery sequence, A6C, and a stem-loop structure in the RNA. The presence of this structure was confirmed by complementary and compensatory mutation analysis and by direct structure probing with single- and double-stranded RNA-specific reagents. The HAst-1 frameshift signal, like that present at the overlap of the gag and pro genes of the retrovirus human T-cell lymphotrophic virus type II, does not involve the formation of an RNA pseudoknot.