To evaluate the most efficient method for preparation of single cell suspensions from viable tumor tissue for use in in vivo experiments, a mouse methylcholanthrene-induced sarcoma and a polyoma virus-induced sarcoma were used. The tumor cells were disassociated by four different methods and the state of viability, yield of single cells per 0.1 gram of tumor, and the quantity of cell debris were determined. The effect on transplantability of each of the methods was assessed by injection s.c. of a constant number of viable cells. Of the four methods tested, the cell preparation resulting from treatment of the tumor with the proteolytic enzyme “Pronase” yielded the highest number of viable cells and the smallest quantity of cell debris. Transplantability of the tumor cell suspension prepared by each of the methods was similar when a constant number of viable cells was injected.