Allo-reactive T cells reactive with Ia antigens from strain 2 and 13 guinea pigs were cloned by plating T cells from primary mixed leukocyte cultures in soft agar and expanding the resulting colonies in liquid culture. Optimal expansion of the cultures from an individual colony was achieved by repeated stimulation with allogeneic cells in conditioned media. Mapping studies using stimulator cells from inbred and outbred guinea pigs and blocking studies with alloantisera and xenogeneic monoclonal antibodies to guinea pig Ia antigens indicated that these T cell colonies were specifically stimulated to proliferate by Ia molecules. In addition, these xenogeneic monoclonal anti-Ia antibodies had selective and differential inhibitory effects on the proliferative response of these alloreactive colonies. This result suggested that each T cell colony is specific for a distinct epitope expressed on Ia molecules. Furthermore, unlike heterogeneous cell populations in guinea pig primary and secondary mixed leukocyte cultures, most alloreactive T cell colonies expressed significant cytotoxic T lymphocyte activity that was also specific for Ia. Thus, the same cell proliferating to Ia is also a T killer cell. These T cell colonies will be useful for further analysis of the specificity and functional significance of the T cells responding in the mixed leukocyte reaction.