Abstract Portions of fresh peribronchial lymph nodes from 92 dogs collected from Loudoun County, Virginia, were homogenized and injected into mice. The mice were sacrificed 4 to 6 weeks after injections, and mouse livers and spleens were cultured for fungi. Direct cultures of peribronchial lymph nodes and 20 or more cultures of other tissues were made on each dog. Histoplasma was isolated from a total of 51 dogs. The organism was isolated from mice receiving peribronchial lymph node homogenates prepared from 48 of the 51 positive dogs. The organism was isolated only by the mouse injection method from 29 dogs. The failure to isolate Histoplasma by direct culture of peribronchial lymph nodes is attributed to the high incidence of contamination of this tissue by saprophytic fungi and occasionally bacteria. In a subsequent series Histoplasma was isolated from mice receiving homogenates prepared from peribronchial lymph nodes of 12 of 19 dogs. The lymph nodes had been stored at −40 °C. for 23 to 160 days before homogenization and injection into mice. The application of the mouse injection method for isolating Histoplasma from clinical specimens is discussed.