The ability of mouse B-mitogen-induced lymphocytes to grow and develop into colonies in a soft agar system was studied. Prerequisite conditions for the colony formation of mouse lymphocytes from inguinal lymph nodes of strains ICR C3H/eB and C3H were their suspension in a liquid medium and stimulation with polyclonal B-cell activators such as bacterial lipopolysaccharide (LPS), purified protein derivative (PPD) or dextran sulphate (DxS) prior to being seeded on a soft agar culture medium. After 3-5 days of culture, colonies of 50-350 cells or more per clone developed. A linear relationship was found between the number of cells seeded and the number of colonies growing. Of the cells seeded, only a limited population of the mitogen-stimulated lymphocytes has the potential to divide and to develop into colonies. The largest number of colonies was obtained by culturing lymph node cells of ICR mice and using LPS as mitogens. Two sublines of C3H were found to respond differently to LPS: C3H/HeJ mice were low responders while C3H/eB mice were high responders. Experiments with inbred, congenitally athymic nude (nu/nu) mice known to be deficient in T cells showed that LPS-stimulated lymphocytes were capable of forming colonies. The morphology of the colony cells, as well as the fact that they stain positively for cell-membrane immunoglobulins, suggest that the colonies developed from B lymphocytes.