Previous studies have established that atopic individuals living in cockroach-infested housing become sensitized to cockroach aeroallergens and produce IgE antibodies to a variety of proteins. We describe the isolation of a complementary DNA clone from an expression library, constructed with messenger RNA from German (Blattella germanica) cockroaches, which encodes a major allergen involved in mediating cockroach hypersensitivity. Approximately 0.2% of the clones from a lambda ZAP XR cDNA library bound IgE from a patient with cockroach sensitivity. A randomly selected subset of these clones revealed that they were either different isolates of the same gene or members of a closely related gene family. One of the largest clones (a 4 kb insert) from this subset, Bla g Bd90K hybridized to a single mRNA of approximately the same size. DNA sequence analysis showed that this gene consisted of seven 576 bp tandem repeats with a short unique region at either end. No significant sequence homologies were found between the cockroach clone and any other gene reported in the GenBank database. Serum from 17 of 22 (77%) patients with cockroach hypersensitivity identified IgE-binding recombinant protein expressed from clone Bla g Bd90K in Escherichia coli XL-Blue cells as determined by sodium dodecylsulfate–polyacrylamide gel electrophoresis/immunoblot analysis. This recombinant protein migrates with a molecular weight (90 kd) apparently similar to one identified in whole body extracts. We have identified and isolated a cDNA that encodes a major cockroach allergen (Bla g Bd90K) present in German cockroaches. (J ALLERGY CLIN IMMUNOL 1996;98:172-80.)