Pancreatic cancer has an extremely poor prognosis and lacks early diagnostic and therapeutic possibilities, mainly because of its silent course and explosive fatal outcome. The histogenesis of the disease and early biochemical and genetic alterations surrounding carcinogenesis are still controversial. In vitro studies offer a useful tool to study physiologic, pathophysiologic, differentiation, and transformation processes of cells and to understand some of these shortcomings. The extreme difficulties in isolating individual pancreatic cells and their purification by maintaining their native characteristics have limited research in this area. This review is intended to present and discuss the current availability of rodent and pancreatic cell lines, their differences as well as the difficulties, limitations, and characteristics of these cultured cells. Discussed are in vitro models; ductal, islet, and acinar cell culture; cell differentiation; cell transformation, including genetic and chromosomal alterations; as well as tumor cell markers. Also addressed are the advantages and problems associated with the cell culture in humans and rodents. Advancements in tissue culture technique and molecular biology offer steady progress in this important line of research. The improved methods not only promise the establishment of beta-cell cultures for the treatment of diabetes, but also for studying sequential genetic alterations during pancreatic carcinogenesis and in understanding the tumor cell origin.