A chemotactic factor extracted from sterile filtrates of Escherichia coli cultures was strongly chemotactic for polymorphonuclear leukocytes (PMN) and rabbit alveolar macrophages (RAM). Electrophoresis of the cytotactic material yielded five lipid fractions: one that was protein-free and active toward both PMN and RAM, and four lipid-protein complexes that were strongly chemotactic only for RAM. Thin-layer chromatography of the lipid-protein complexes resulted in an unmasking of PMN activity in a peptide-free lipid extract, while the isolated peptidic components were essentially noncytotactic. The original RAM activity was retained in the unmasked lipid, which possessed chemical and chromatographic properties similar to those of a previously reported cytotaxin synthesized from arachidonic acid. These data indicate that a class of lipids derived from bacterial and cellular sources is intrinsically cytotactic for PMN and RAM. When peptide moieties are associated with cytotactic lipids, the resultant lipid-peptide complex may exhibit cellular specificity not evident in the free lipid.