Normal mesenchymal cells, normal epithelial cells and many transformed epithelial cells require serum attachment factors and extracellular matrix proteins for growth and differentiation in vitro, and recent evidence strongly supports a role for extracellular matrix molecules in the regulation of cell movement in vivo during early embryogenesis. We previously described the isolation and characterization of cell lines representative of three types of stem cells most commonly found in human adult testicular teratomas, namely embryonal carcinoma cells, yolk sac carcinoma cells resembling visceral endoderm and yolk sac carcinoma cells resembling parietal endoderm (endodermal sinus tumour cells). Of these three cell types, only endodermal sinus tumour cells, which show particularly malignant behaviour in vivo, have no serum requirement for attachment and growth in vitro. Supernatants from endodermal sinus tumour cells support the attachment of embryonal carcinoma cells in serum-free medium. We demonstrate here that endodermal sinus tumour cells, but not other cell types isolated from testicular teratomas, secrete the serum attachment protein, vitronectin (also known as serum-spreading factor, S-protein or epibolin), as well as fibronectin, laminin and type IV collagen, into serum-free medium. Purified vitronectin from medium conditioned by endodermal sinus tumour cells supported both attachment and spreading of embryonal carcinoma cells in vitro, whereas cells attached but did not spread properly on surfaces coated with fibronectin or laminin. Peptides containing the RGD cell recognition sequence common to many attachment proteins blocked attachment of endodermal sinus tumour cells to untreated tissue-culture plastic in serum-free medium. The results suggest a possible role for vitronectin in regulating cell motility and growth in early development, and in the invasion and spread of teratomas in vivo.