Incubation of murine thymocytes with thymosin Fraction 5 (F5) results in a twofold enhancement of the cytotoxic T lymphocyte response (CTL). The assay exhibits requirements for optimal concentrations of thymosin (100 micrograms/ml) and optimal responder/stimulator ratios. Enhancement of CTL activity can be demonstrated in several responder/stimulator strain combinations. The data indicate that thymosin F5 acts via the responder thymocyte population rather than the stimulator cells, since comparable effects were obtained using nude spleen stimulator cells devoid of mature T cells. This system provides a useful bioassay for identifying the component peptides of thymosin F5 which promote thymocyte differentiation and/or maturation and for elucidating the mechanisms of action of the biologically active thymosin peptides.