Sporothrix schenckii cell wall peptido-rhamnomannan (CWPR) was fractionated by affinity chromatography on Sepharose 4B-Concanavalin A giving rise to S. schenckii concanavalin A-binding (SsCBF) and nonbinding (SsNBF) fractions. The CWPR, SsCBF and SsNBF fractions were probed by enzyme-linked immunosorbent assay (ELISA) with the sera of 35 patients with sporotrichosis from Latin America. Both CWPR and SsNBF, although reacting with sera from patients with sporotrichosis, were also cross-reactive with sera from healthy individuals and from patients with other mycoses. In contrast, SsCBF was shown to react specifically with 100% of the sera from patients with sporotrichosis, compared with control sera and sera from patients with other mycoses or cutaneous leishmaniasis. This antigenic fraction was submitted to mild alkaline hydrolysis to remove O-glycosidically linked oligosaccharides and the resulting components had decreased reactivity with patients' sera. The purified SsCBF-derived O-linked pentasaccharide and tetrasaccharide were potent inhibitors of, respectively 76 and 53% of the reaction between SsCBF and sera from patients with sporotrichosis. Our results suggest that SsCBF is a species-specific antigenic fraction recognized by human sera and that this specificity correlates with the presence of O-linked mannose-containing oligosaccharides decorated with the alpha-L-Rha 1-->4 alpha-D-GlcA and alpha-L-Rha 1-->4 [alpha-L-Rha 1-->2] alpha-D-GlcA epitope structures previously described [1].