The genome of tomato golden mosaic virus (TGMV) is composed of two circular, single-stranded DNA molecules that together contain 6 open reading frames (ORFs). Three of these ORFs (designated AL1, AL2, and AL3) overlap and are specified by multiple polycistronic mRNAs. No RNA specifying the AL3 ORF alone has been detected, suggesting that the AL3 gene product is translated from an internal ORF. A recombinant histidine-tagged-AL3 fusion protein was purified from Escherichia coli and used to raise a polyclonal antiserum. Analysis of protein extracts from healthy plants and plants infected with TGMV by SDS-PAGE and immunoblotting showed that a protein corresponding to the predicted AL3 gene product is produced only in infected plants. This protein comprises approximately 0.05% of the cellular proteins and is present in the soluble and organelle fractions. These results are discussed with respect to the expression and role of the AL3 protein in the viral life cycle.