A cell motility-stimulating factor has been isolated, purified, and partially characterized from the serumfree 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 p transforming growth factor.