AbstractAutocrine motility factor (AMF) is a cytokine that regulates locomotion and metastasis of tumor cells. It is well known that expression levels of AMF secretion and its receptor (AMF R) are closely related to tumor malignancy and rheumatoid arthritis. We have established that AMF signaling induced anti‐apoptotic activity and that human fibrosarcoma HT‐1080 line that secreted high levels of AMF were resistant to drug‐induced apoptosis. These cells did not express the apoptotic protease activating factor‐1 (Apaf‐1) and Caspase‐9 genes that encode for the proteins that form the “apoptosome” complex. The disappearance of the Apaf‐1 and Caspase‐9 gene was recovered by a cellular signaling inhibitor of protein kinase C, phosphatidylinositol 3‐phosphate kinase and mitogen‐activated protein kinase of the in vitro cultured human fibrosarcoma HT‐1080 line. Treatment with these inhibitors favored apoptotic cell death induced by anti‐cancer drugs of the murine ascites Ehrlich line. Apoptotic resistance of tumor cells allows them to escape death from cancer chemotherapy, so an understanding of malignant anti‐apoptotic activities is important. Antibodies against AMF induced Ehrlich ascites apoptosis in vitro, and effectively aided in vivo apoptosis induced by anti‐cancer drugs. The results might indicate a novel route by which tumor cells protect themselves with products, such as AMF, and proliferate despite various stresses and chemical insults; AMF regulates expression of Apaf‐1 and caspase‐9 genes via a complex signaling pathway and indirectly regulates formation of the apoptosome. © 2003 Wiley‐Liss, Inc.