Sixty-four kinds of cell lines were examined for their ability to produce megakaryocyte potentiating activity by means of conditioned media obtained from a protein-free culture system. Six human tumor cell lines were shown to produce this activity, and the cell line HPC-Y5, established from human pancreatic cancer, was shown to have the highest level of activity. The megakaryocyte potentiating factor (MPF) was purified from an HPC-Y5 conditioned medium by a combination of ion-exchange chromatography, gel filtration and reverse-phase HPLC. The purified MPF showed a megakaryocyte potentiating activity almost equal to human interleukin-6 in the presence of murine interleukin-3 in a colony formation assay with mouse bone marrow cells. The apparent molecular weight of MPF is 32,000 when determined by SDS-polyacrylamide gel electrophoresis. Glycopeptidase F digestion, and amino sugar analysis of the factor demonstrated that MPF is a glycoprotein carrying at least one N-linked sugar chain. The N-terminal amino acid sequence of MPF was determined to be Leu-Ala-Gly-Glu-Thr-Gly-Gln-Glu-Ala-Ala-Pro-Leu- Asp-Gly-Val-Leu-Ala-Asn. The same or homologous amino acid sequence has not been found in known proteins, demonstrating that MPF is a novel cytokine that has megakaryocyte potentiating activity in the murine assay system.