A technique for the multiple transformation of yeast by protoplast fusion is described. This involved the PEG-induced fusion of protoplasts from cells which had been treated with chromosome-fragmenting agents (in this case cupferron and hydroxylamine) with protoplasts of triply auxotrophic cells. The recovery of transformants was increased significantly if one of the amino acid requirements of the recipient strain was included in the selection medium. Transformants isolated on supplemented media remained auxotrophic for that requirement. Prototrophic, uninucleate transformants had a DNA content and cellular volume similar to that of the parental strains. Possible mechanisms of gene transfer are discussed. This technique offers the possibility of transferring desirable characteristics from one yeast strain to another without altering the ploidy level of the recipient strain.