Dermatophytes of the genus Trichophyton cause infections of human skin, nails, and hair. Unlike most Ags, Trichophyton can elicit either immediate (IH) or delayed (DH) hypersensitivity skin reactions. Previous studies isolated a 30-kDa Ag (Tri t 1) that caused IH skin tests. The study presented here used skin testing and in vitro T cell proliferation assays to monitor purification of an Ag, designated Protein IV, associated with DH reactions. Protein IV was purified by cation exchange HPLC; amino acid sequence analysis of the N-terminus and nine internal peptides (143 residues) revealed no homologies to Tri t 1 or to any other known proteins. A mAb-based ELISA was developed to measure Protein IV. Protein IV elicited DH skin reactions in subjects with a history of athlete's foot but also caused IH skin reactions. Serologic responses to Protein IV were studied in 59 adults who had been skin tested with Trichophyton extract. IH skin reactions were associated with a positive RAST (14/23) as well as with specific IgE (13/23) and IgG4 (14/23) Abs to Protein IV. DH skin tests were not associated with IgE or IgG4 Abs. IgE anti-Protein IV Abs were quantitatively correlated with IgG4 Abs (r = 0.57, p < 0.001). Specific IgG Abs to Protein IV were highest in IH subjects (gm = 230 U/ml), and lowest in those with DH (gm = 91 U/ml) or negative (gm = 81 U/ml) skin tests; furthermore, the prevalence of IgG Abs increased significantly with age. Protein IV is the first defined protein associated with both DH and IH skin reactions; these reactions are characterized by distinct serologic responses. The results establish that diverse immune responses in humans can be directed against the same protein.