Spleen lymphocyte proliferation, as measured by 3H-thymidine incorporation induced by phytohaemagglutinin (PHA), was increased after freeze-thawing with 10% dimethyl sulphoxide. Depletion or intoxication of macrophages in fresh spleen cell preparations also increased lymphocyte proliferation in response to PHA. On the other hand, freezing of macrophage-depleted spleen cell suspensions lowered 3H-thymidine uptake of stimulated cultures. At concntrations above 3%, macrophages added to cultures of fresh purified lymphocytes showed a dose-dependent inhibitory effect on the PHA response, and fresh macrophages were more inhibitory than frozen-thawed macrophages. Purified lymphocytes mixed with 10% macrophages showed a higher response after freeze-thawing. It is concluded that macrophages suppress the lymphoproliferative response to PHA in rat spleen cell cultures, and that these macrophages are more sensitive than lymphocytes to the present freeze-thaw process.