SUMMARY The use of frozen stored leukocytes (FSL) for studies of in vitro cell-associated immune responses to specific antigens, allogeneic cells, and phytohemagglutinin (PHA) are described. Previous attempts to restore in vitro reactivity of FSL to specific antigens or to frozen allogeneic lymphocytes in the mixed lymphocyte reaction were unsuccessful despite the fact that frozen cells responded well to PHA and to fresh allogeneic cells. In this study, we have found that fresh human plasma (FHP) or serum restored the ability of frozen cells to respond to antigen and frozen allogeneic cells. Morphological and kinetic analysis indicated that FSL respond in a manner similar to fresh cells. Moreover, leukocytes obtained from the same subject at different periods of time and stored in the frozen state were found, if cultured at the same moment, to respond in a quantitatively reproducible manner to specific antigen, PHA, or allogeneic cells. Such results held true for FSL stored for as long as 9 months. The use of FSL thus adds a new dimension to the quantitative in vitro studies of cell-associated immunity.