Epidemiological studies of paracoccidioidomycosis have been based on surveys achieved with intradermal tests, and paracoccidioidin is the most common antigen used in most cases. The glycoprotein of 43-kDa (gp43) has been used in intradermal tests. It is the most antigenic component of Paracoccidioides brasiliensis, and it provides greater specificity to evaluate infection for this fungus. In this study, the prevalence of P. brasiliensis infection was estimated with intradermal tests involving gp43 for 695 people in rural Central-West Brazil. The infection rate was 45.8 % (95 % CI = 42.1-49.5), and the average age of those infected was 45.8 ± 18.2 years. The prevalence did not show gender-based differences but increased with age. The results demonstrate the importance of P. brasiliensis infection in rural settlements and the early exposure of children in the region to the fungus. Despite the high antigenicity and specificity of gp43, its usage must be standardized, so that epidemiological surveys will be comparable and more accurately reflect P. brasiliensis infection in endemic areas.