A cell-free growth medium for the opportunistic pathogenic ameba Balamuthia mandrillaris is presented. This represents an advance over the use of monkey kidney cells for growth of the amebas and can be helpful in isolation of these amebas from brain tissue from cases in which amebic meningoencephalitis is a diagnostic possibility, as well as for biochemical and molecular biological studies. Three isolates of Balamuthia have been cultured in this medium. The cell-free growth system was also used to screen cultures for sensitivity to a variety of antimicrobial agents. Of the various drugs tested, pentamidine isethionate was most effective against amebas (ca. 90% inhibition after 6 days of exposure), but the drug was amebastatic and not amebacidal in the axenic system at the highest concentration tested (10 micrograms/ml).