A purified glycoprotein of 43,000 daltons from Paracoccidioides brasiliensis (gp43) was tested as paracoccidioidin in delayed-type hypersensitivity (DTH) tests in both experimental animals (guinea pig and mice) and patients with paracoccidioidomycosis (PCM). The gp43 paracoccidioidin was compared with the traditional Fava Netto antigen (AgFN). In guinea pigs, the intradermal injection of 2 micrograms of gp43 showed a similar response to those obtained with AgFN, showing in histological sections a population of lymphoid cells that participate in DTH. In mice, gp43 at a dose of 3.75 micrograms showed positive DTH response. The use of gp43 as paracoccidioidin in humans showed that this molecule can be used to evaluate the DTH response in patients with PCM. Of 25 PCM patients studied, 48% were positive to gp43 while only 28% were positive to AgFN; 12 PCM patients were completely anergic to both antigens. Considering only those 13 PCM patients who were responsive to gp43 and/or to AgFN, 92.3% reacted against gp43 and 53.8% reacted against AgFN (P < 0.05). Gp43 skin test responses (13.67 +/- 9.56 mm) were significantly larger than those obtained with AgFN (8.43 +/- 3.69 mm). Immunohistochemical study of the human skin showed a perivascular inflammatory response constituted predominantly by T lymphocytes, macrophages and polymorphonuclear leukocytes.