Two additional members of the American cockroach (Periplaneta americana) Per a 3 (Cr-PI) allergen, C13 and C28, were isolated and sequenced. They encoded proteins of 470 and 393 amino acids with two and no potential N-glycosylation sites, respectively. The molecular weights for C13 and C28 cloned proteins are 56,200 and 46,7000, with PI values of 7.06 and 6.54. C13 and C28 display 95.4% identity with several overlapping predicted central antigenic determinants. Both allergens were also found to have a 95% sequence homology with previously cloned C20 and share similar antigenic determinants, as defined by the structural prediction and ELISA analysis. However, the recombinant C13 and C28 allergens showed 26.3 and 94.7% skin reactivities on asthmatic patients while C20 elicited 47.4%. While no sequence similarity was found to other known allergens, these two aromatic amino acid-rich allergens were highly related to insect hemolymph proteins (28.7-36.5%), as with C20 cloned protein. Results suggest that these two are isoallergenic variants of C20. Sequence variations among isoforms, resulting a significant difference in skin reactivities, will be useful in elucidating the allergenic determinants.