RNA 3 of alfalfa mosaic virus encodes the movement protein P3 and the viral coat protein (CP). CP is translated from a subgenomic (sg) messenger, RNA 4. To characterize the sg promoter that is responsible for RNA 4 synthesis in vivo, putative sg promoter sequences were inserted in a unique Xhol site located between the initiation codon of the P3 gene and a second in-frame ATG codon in an infectious cDNA clone of RNA 3. Mutants with an active sg promoter insert expressed an N-terminally truncated P3 protein and were able to accumulate in plants. In addition, sg promoter activity was analyzed in protoplasts. When the transcription start site is taken as +1, the sequence of nucleotides -26/+1 was found to have a basal level of sg promoter activity. This activity was increased to near maximum levels when the sg promoter sequence was extended to -136/+12. The upstream positive regulatory element was mapped to nucleotides -136/-94. Engineering of point mutations and small deletions in RNA 3 around the transcription start site for RNA 4 synthesis revealed elements important for sg promoter activity with similarity to sequences conserved in sg promoters of alpha-like viruses. Some of these elements appeared to be required in cis for RNA 3 accumulation. A deletion of the C-terminal three amino acids of the P3 protein rendered this protein nonfunctional in cell-to-cell movement.