The binding of the mitogenic lectins concanavalin A, Wistaria floribunda mitogen, Pisum sativum hemagglutinin, Lens culinaris lectin, phytohemagglutinin, and two of the pokeweed mitogens (Pa-1 and Pa-2), in mitogenic concentrations to the cell surface acceptors of murine splenic lymphocytes caused a rapid, transient increase in the methylation of lymphocyte phospholipid. The extent of this increase was dependent upon the concentration of the lectin, and paralleled the dose-response curve for subsequent mitosis. Higher or lower doses of lectin did not cause the observed changes in methylation nor induce mitosis. The increase of phospholipid methylation reached a maximum about 10 min after the addition of the mitogenic lectins and then decreased to control levels within about 45 min. Accordingly, mitogenic lectins not only stimulate phospholipid methylation in lymphocytes, but also stimulate degradation of the phospholipids, probably by phospholipase A2. The non-mitogenic lectins, Sophora japonica hemagglutinins (Sj-I and Sj-II), W. floribunda hemagglutinin, and Bauhinea purpurea hemagglutinin did not significantly affect the phospholipid methylation. Preincubation of lymphocytes for 1 hr with 10 −5 M of the methylation inhibitor. S-isobutyryl-3-deazaadenosine, suppressed both the concanavalin A (Con A)-induced increase of phospholipid methylation after 10 min, and subsequent DNA synthesis after 45 hr. This concentration of methylation inhibidid not affect these responses if added immediately after the Con A stimulation, and appears to be specific for the phospholipid methylation. The degradation of methylated phospholipid was blocked by the phospholipase A2 inhibitor, mepacrine, which also suppressed DNA synthesis. The T-cell mitogen, Con A. showed the same lymphocyte specificity for phospholipid methylation enhancement as for stimulation of DNA synthesis. Con A caused the transient increase of phospholipid methylation in murine thymocytes, spleen cells treated with anti-immunoglobulin and complement, and purified T-cells. On the other hand, Con A did not cause a meaningful change of phospholipid methylation in spleen lymphocytes from athymic nude mice, spleen cells treated with rabbit anti-mouse brain and complement, or purified B-cells. From these results, it appears that both increased phospholipid methylation and degradation in mitogen-stimulated lymphocytes may be an integral part of the mitogenic signal.