A lambda gt11 library made with cDNA from the house dust mite Dermatophagoides pteronyssinus was screened with human allergic serum by IgE plaque radioimmunoassay. This resulted in the isolation of clones coding for the major allergen Der p II. The cDNA coded for a 129-residue protein of 14,131 daltons with no N-glycosylation sites. No sequence homology with other proteins was evident. The Der p II expressed in Escherichia coli reacted with IgE in 14 of 17 sera from mite-allergic patients giving clonal evidence for its designation as a major allergen. This, along with previous work, has resulted in the cloning of the two major mite allergens.