A cell motility-stimulating factor has been isolated, purified, and partially characterized from the serum-free conditioned medium of human A2058 melanoma cells. We term this activity "autocrine motility factor" (AMF). AMF has the properties of a protein with an estimated size of 55 kDa. At concentrations of 10 nM or less, AMF stimulated the random or directed motility of the producer cells. However, AMF is not an attractant for neutrophils. Amino acid analysis of the purified AMF protein revealed a high content of serine, glycine, glutamic acid, and aspartic acid residues. The activity of AMF was not replaced or blocked by known growth factors such as epidermal growth factor or type beta transforming growth factor. Mechanistic studies showed that AMF stimulated the incorporation of [3H]methyl into cell membrane phospholipids after incubation with [methyl-3H]methionine with a sustained increase in the methylation of phosphatidyldimethylethanolamine to phosphatidylcholine. In contrast, AMF did not affect the incorporation of [1,2-14C]choline into phosphatidylcholine. AMF was produced in large amounts by three different clones of ras oncogene-transfected metastatic NIH 3T3 cells but not by the nontransformed parental cells. AMF may play a major role in the local invasive behavior of tumor cells and may also facilitate the concerted invasion by groups of tumor cells.