This report describes the first isolation of the ameba Balamuthia mandrillaris from an environmental soil sample associated with a fatal case of amebic encephalitis in a northern California child. Isolation of the ameba into culture from autopsied brain tissue confirmed the presence of Balamuthia: In trying to locate a possible source of infection, soil and water samples from the child's home and play areas were examined for the presence of Balamuthia: The environmental samples (plated onto nonnutrient agar with Escherichia coli as a food source) contained, in addition to the ameba, a variety of soil organisms, including other amebas, ciliates, fungi, and nematodes, as contaminants. Presumptive Balamuthia amebas were recognized only after cultures had been kept for several weeks, after they had burrowed into the agar. These were transferred through a succession of nonnutrient agar plates to eliminate fungal and other contaminants. In subsequent transfers, axenic Naegleria amebas and, later, tissue cultures (monkey kidney cells) served as the food source. Finally, the amebas were transferred to cell-free axenic medium. In vitro, the Balamuthia isolate is a slow-growing organism with a generation time of approximately 30 h and produces populations of approximately 2 x 10(5) amebas per ml. It was confirmed as Balamuthia by indirect immunofluorescence staining with rabbit anti-Balamuthia serum and human anti-Balamuthia antibody-containing serum from the amebic encephalitis patient. The environmental isolate is similar in its antimicrobial sensitivities and identical in its 16S ribosomal DNA sequences to the Balamuthia isolate from the deceased patient.