During the construction of yeast artificial chromosome (YAC) libraries to facilitate mapping of the human genome, two YACs may be cotransformed into the same yeast cell, making further analysis very difficult. We present a simple method to rescue the required YAC that utilizes the segregation of chromosomes at meiosis. In brief, we crossed the cotransformed yeast cell with a non-YAC-containing strain and induced the resulting diploid to sporulate and undergo meiosis. The new haploid generation included some yeast cells that contained only the desired YAC. These YACs were analyzed by conventional methods. To exclude the possibility that major rearrangement occurred during the procedure, we analyzed the YACs with restriction enzymes that cut only rarely. We conclude that this is a useful technique to rescue cotransformed YACs. © 1993.