In order to improve the methods for the cytogenetic analysis of small rodents with regard to metaphase number, quality of chromosome resolution and duration of cultivation, a short-term culture technique from spleen cells, based on the capacity of Concanavalin A supernatant to stimulate the proliferation of normal T lymphocytes, was developed. Protocols of culture and chromosome preparation are described in detail. This technique not only makes it possible to obtain a high number of cells in mitosis from the spleen of very small rodents in a short time, but also insures metaphases of good quality, due to optimal control of cell cycle and processing thanks to cultivation in small aliquots. Although the method applies mainly to rodents because of species specificity of interleukin 2, it can probably be easily adapted to species from other mammalian orders.