Cell-mediated immunity is critical in host resistance against the pathogenic fungus Histoplasma capsulatum. To explore the role of L3T4+ T cells in protection of mice against H. capsulatum infection, we examined the effect of in vivo treatment with anti-L3T4 monoclonal antibody (MAb) GK1.5 on the course of murine disseminated histoplasmosis. Treatment with anti-L3T4 antibody caused a profound and selective depletion of L3T4+ T cells that was associated with a significant increase in the number of H. capsulatum CFU recovered from the spleens of mice infected for 1 week. In addition, none of the infected mice treated with MAb GK1.5 survived a sublethal challenge with H. capsulatum yeasts. Histopathological examination of spleens from mice infected for 1 week revealed the presence of granulomatous inflammation in mice depleted of L3T4+ T cells and in infected controls. However, silver stains demonstrated that spleens of infected mice given MAb GK1.5 contained a greater number of yeasts than did spleens from infected controls. MAb GK1.5 did not cause reactivation of infection when administered for 2 weeks beginning 4 weeks after inoculation of Histoplasma yeasts. MAb GK1.5 did not alter the functional properties of murine macrophages as measured by antigen presentation, production of interleukin-1 in response to lipopolysaccharide, and phagocytosis of H. capsulatum yeasts. These results suggest that the L3T4+ T-cell subset is an essential constituent of the cell-mediated immune defense against H. capsulatum infection.