Isolating a stable clone of hybridoma cells that all secrete the correct antibody is the most time-consuming step in the production of hybridomas. Single-cell cloning ensures that cells that produce the antibody of interest are truly monoclonal and that the secretion of this antibody can be stably maintained. The original positive well will often contain more than one clone of hybridoma cells, and many hybrid cells have an unstable assortment of chromosomes. Both of these problems may lead to the desired cells being outgrown by cells that are not producing the antibody of interest. Cloning hybridoma cells by limiting dilution is the easiest of the single-cell-cloning techniques. Two approaches are presented here, one rapid technique for generating cultures that are close to being single-cell-cloned and one for single-cell cloning directly. Even though every attempt is made to ensure that the cells are in a single-cell suspension before plating, there is no way to guarantee that the colonies do not arise from two cells that were stuck together. Therefore, limiting dilution cloning should be performed at least twice to generate a clonal population.