An experimental study was conducted between January 2002 and April 2003 for the detection of delayed hypersensitivity to Fonsecaea pedrosoi metabolic antigen (chromomycin) in skin tests. A total of 194 subjects were attended by spontaneous demand at the Infectious and Parasitic Diseases outpatient clinic of the Federal University of Maranhão-UFMA and at the Department of Microbiology, Federal University of Minas Gerais-UFMG and classified into three groups: patients with chromoblastomycosis caused by F. pedrosoi (n=20), healthy subjects (n=86) and patients with other diseases (n=88). For the skin test, 0.1 ml of the antigen was applied to the anterior side of the right forearm and 0.1 ml Smith medium was applied to the anterior side of the left forearm as control. The results were analyzed 48 h after inoculation of the antigen and an induration >/= 5 mm was considered to indicate a positive test. A cellular immune response to chromomycin was detected in 18 (90.0%) of the 20 patients with chromoblastomycosis caused by F. pedrosoi, and one of the patients with a negative test had reactional leprosy. Eighty-five (98.8%) of the 86 healthy subjects presented a negative reaction and only one reacted positively to the antigen. The skin test was negative in all 88 (100%) patients with other diseases, such as dermatophytosis, paracoccidioidomycosis, pulmonary aspergilloma, candidiasis, pityriasis versicolor, tuberculosis, leprosy, tegumentary leishmaniasis and syphilis, and one case of chromoblastomycosis caused by Rhinocladiella aquaspersa. Chromomycin was effective in detecting delayed hypersensitivity in patients with chromoblastomycosis caused by F. pedrosoi, with a sensitivity and specificity of 90.0% and 98.8%, respectively. These results suggest that this antigen can be used in the auxiliary diagnosis of the disease and also in epidemiological studies for determination of the prevalence of chromoblastomycosis infection in endemic areas.