By using ethylene glycol extraction of whole submerged cultures followed by Sephadex G-200 and diethylaminoethyl-Sephadex chromatography, we isolated four distinct glycopeptides from Trichophyton mentagrophytes, T. rubrum, and Microsporum canis. Chemical analyses revealed that these glycopeptides contained mostly carbohydrate (42.5 to 81.6%) and protein (4.3 to 11.3%), with lesser amounts of phosphorus (0.4 to 6.0%) and hexosamines (0.3 to 0.6%). Based upon total carbohydrate and monosaccharide content, these dermatophyte glycopeptides could be divided into two chemical groups: glucopeptides (DSI1) and mannopeptides (DSI2, DSII1, and DSII2). The mannopeptides and glucopeptides of each species of dermatophyte were not significantly different chemically from those derived from the other two dermatophyte species studied. Skin testing of DSI1-glycopeptides or DSI2-mannopeptides in immunized guinea pigs indicated that only the DSI2-mannopeptides elicited a delayed hypersensitivity reaction. Skin testing T. mentagrophytes 62-infected guinea pigs with the four purified DS-glycopeptides, as well as earlier fractions from the purification scheme, derived from T. mentagrophytes, T. rubrum, and M. canis, again indicated that only the DSI2-mannopeptides of the two Trichophyton species elicited a delayed hypersensitivity reaction. The number of infections or duration of infection had no effect on the size of the skin test response. DSI2-mannopeptides were non-cross-reactive between genera when tested in Trichophyton-immunized or -infected guinea pigs and Microsporum-immunized guinea pigs.