NPC-N cells were normal human nasopharyngeal epithelial cells transformed by transfection with human papillomavirus type 16 deoxyribonucleic acid. Bovine pituitary extract (BPE) was one of the indispensable ingredients for in vitro culture of NPC-N cells in a serum-free medium. Chromatographic fractionation of BPE and subsequent immunoblotting analyses identified the hemoglobin growth-stimulating factor. Methemoglobin (metHb) was then synthesized, and also found to be growth stimulating. The growth-stimulating effect of metHb was abolished when NPC-N cells were cultured in a medium that also contained haptoglobin, a molecule that binds to hemoglobin. A defined medium consisting of insulin and metHb was then developed for optimal growth of NPC-N cells. MetHb kept under the conditions identical to those of cell culture released hemin which also enhanced the cell growth. Though all the degradation products of hemin are currently known to be physiologically significant, only ferric iron derived from metHb or hemin could stimulate the growth of NPC-N cells. Abnormal vasculature showing leaky walls and hemorrhage is a common feature of malignant tumors. Hemoglobin originating from extravasated red blood cells and subsequently oxidized to metHb because of the presence of activated inflammatory cells might contribute to the increased proliferation of cancerous cells.