The proliferative ability of fat cells has been studied both in the adipose tissue in vivo and in culture. We studied the proliferation of unilocular fat cells of young rats in a threedimensional collagen gel matrix culture, which provides a physiological environment for fat cells. In this setting, the unilocular fat cells were able to maintain their cellular functions and actively proliferate. In this study using cells derived from 3 to 6-week-old rats, we observed the appearance of very small fat cells at the surface of unilocular fat cells following the division of the nucleus. We concluded that small fat cells were created mostly in a “budding” manner, and then proliferated. Insulin accelerated both the budding and proliferation process. In other unilocular fat cells, lipid droplets divided after the division of the nucleus, and very small fat cells appeared within the unilocular fat cells in a “sharing” manner. Adenosine deaminase, a potent lipolytic factor, accelerated this process. An analysis of the cellular volume by a Coulter counter confirmed the proliferation of small fat cells derived from the unilocular fat cells. These small fat cells grew and developed into unilocular fat cells. It is supposed that not all unilocular fat cells are terminally differentiated, nor do they lose their proliferative activity.