Chemotaxis by human monocytes has been shown to require methylation mediated by S-adenosyl-L-methionine(AdoMet), but the specific transmethylation reaction necessary for this function was not elucidated. In an attempt to define the methylation requirement for chemotaxis, we examined the effect of chemotactic agonists and antagonists on protein carboxy-O-methylation of protein and methylation of phospholipid in guinea pig macrophages. Chemotactic agents tested over a wide dose and time range produced no alteration in carboxy-O-methylation. However, these agents did produce an effect on the methylation of phosphatidylethanolamine by macrophages. AdoMet-mediated phospholipid methylation was inhibited by as much as 73% by chemotactic factors, and there was excellent correlation (r = 0.99) between their concentrations for producing half-maximal chemotactic responses and for inhibiting phospholipid methylation. The inhibition of methylation by chemotactic factors was observed at all incubation times and could not be explained by an increased turnover of membrane phospholipid. Neither the chemotaxis antagonist fPhe-Met nor the nonchemotactic tripeptide Met-Met-Met significantly depressed phospholipid methylation. Immune phagocytosis by macrophages similarly did not alter phospholipid methylation. The chemotactic factors produced no alteration in total macrophage phospholipid synthesis or in the phospholipid methylation in a nonchemotactic cell type. The formation of newly methylated derivatives of phosphatidylethanolamine in macrophages was decreased by a biologically active dose of chemotactic factor. These findings indicate that chemotactic factors are capable of altering the methylation of phosphatidylethanolamine in chemotactically responsive cells. The inhibition of phospholipid methylation by chemotactic factors may be necessary for the translation of a chemotactic signal on the surface of the cell into directional cell movement.