Human blood leucocytes from certain individuals cultured aloneas "controls" for experiments designed to demonstrate the mutual reactivity of mixed cultures of leucocytes from different donorsrevealed an intense proliferative response by the Wth day as measured by the incorporation of radioactive thymidine. Furthermore, thymidine incorporation into control leucocyte cultures from most donors was markedly elevated by the seventh day of culture. Because these high background levels tended to obscure the mutual reactivity of mixed leucocyte cultures, experiments were designed to elucidate the nature of and to circumvent these difficulties. By supplementing the growth media with human plasma, rather than calf serum, the background activity of the control cultures could be maintained at low levels without compromising the mutual reactivity of lymphocytes in the nixed cultures. One other source of variability which proved simple to control was the relative number of "contaminating" non-lymphoid cells in the cultures. This factor was important since, in the preparation of the leucocyte suspensions with erythrocyte sedimentation procedures, the number of contaminating granulocyte cell types can be expected to vary over a wide range.
Phytohemagglutinin (PHA) was used as a mitogen in experiments designed to define further some of the parameters of the lymphocyte response i n vitro. When a standard number of lymphocyteseither as a "purified" suspension or as one containing significant numbers of contaminating granulocyteswas cultured with various concentrations of PHA, a dose-dependent response was observed. Partially purified lymphocyte suspensions were only one-half to one-third as active as non-purified leucocyte suspensions containing the aame number of lymphocytes. This could not be attributed to a deleterious effect of PHA on purified cultures with a lower cell density since (1 ) the peak response of both cultures occurred with the same concentration of PHA, and (2) the maximal reactivity to PHA could be restored by increasing the granulocyte content.