A new human pancreatic cancer cell line, designated YPK-1, was established from ascites collected from a 62-year-old Japanese man. This cell line has been maintained in vitro for 96 months with stable growth through 260 passages. We characterized the YPK-1 cell line morphology, doubling time, levels of tumor markers secreted into culture medium, chromosomal content, and tumorigenicity in nude mice. YPK-1 cells have a clear polygonal-shaped cytoplasm, round nuclei, and prominent nucleoli. The cells grew as an adherent monlayer and lacked contact inhibition. The doubling time at the tenth passage was 49.3 hours. YPK-1 cells secreted detectable amounts of carcinoembryonic antigen, carbohydrate antigen 19-9, carbohydrate antigen 125, carbohydrate antigen 50, DU-PAN-2, and Span-1. Chromosome analysis revealed that the diploid chromosome number ranged from 57 to 62 with a mode of 61. Tumorigenicity was demonstrated by development of tumors after subcutaneous injection of YPK-1 cells into nude mice. We believe that this cell line will contribute to studies of the biological and molecular properties of pancreatic carcinoma and the development of therapeutic strategies for this tumor.