We have cloned and sequenced Amb a V, an Ambrosia artemisiifolia (short ragweed) pollen allergen that has proved to be particularly useful in the genetic analysis of human immune responsiveness. The amino acid sequence deduced from the cloned cDNA sequence corresponds to the published sequence of the protein, except that the cDNA sequence encodes an extra 10 amino acids at the C-terminus. The expressed 55-residue protein is then presumably cleaved enzymatically at the C-terminal lysine found in the 45-residue protein isolated from the pollen. The cloning and sequencing of Amb a V genomic DNA confirmed the cDNA sequence and showed that the Amb a V gene has no introns. Recombinant Amb a V allergen, expressed in Escherichia coli, bound to IgG and IgE antibodies in all Amb a V-allergic individuals tested and inhibition studies demonstrated that the recombinant protein contains a subset of the antigenic epitopes found on native Amb a V. In addition, recombinant Amb a V released histamine efficiently from basophils from Amb a V-allergic patients. The recombinant Amb a V allergen and mutants of Amb a V should, therefore, be useful in studies of allergen epitopes in humans, as well as providing a diagnostic tool.