Cholesterol auxotrophy of myeloma cells is used as the basis for selecting hybridomas. The outgrowth of nascent hybridomas in a cholesterol-free medium was 3- to 9-fold more efficient than that in HAT medium and resulted in 3- to 13-times as many antigen-reactive hybridoma cells. This method of hybridoma selection can be applied with any sterol-dependent parent cell line. The nutrient medium is also preferably free of Ham's F-12 nutrient mixture.