To determine the number of adipocytes and cells developing into adipocytes (preadipocytes) in the epididymal fat pad of normal Sprague-Dawley rats, two methods were developed. Liberation of all cells from the tissue was obtained by a combination of lytic enzymes and mechanical treatment with only a limited loss of cell integrity; with large tissue masses, an initial perfusion was necessary. These cells were cultured in medium 199 supplemented with serum, glucose, insulin, a triglyceride emulsion, and methyl cellulose to form a culture medium with high viscosity in which it has been shown that the cells do no multiply. In this medium some of the cells developed into adipocytes and could be recognized and counted. The results show that there are about twice as many preadipocytes as mature adipocytes in the smallest rats examined (about 50 g). With increasing weight and age the mature adipocytes increased while the number of preadipocytes seemed to be constant up to a weight of about 150 g, after which they continuously diminished and could not be found in rats weighing more than 300 g. Here the number of mature fat cells had reached a constant level. These results are consistent with the formation of new preadipocytes up to a rat weight of about 150 g. In normal rats these cells successively fill up with triglyceride and disappear at a body weight of about 300 g when they have been transformed into mature adipocytes. The results are also consistent with the concept that no new adipocytes are formed spontaneously in the adult Sprague-Dawley rat. It was shown, however, that in media without methyl cellulose, isobutylmethylxanthine probably could induce the formation of new adipocytes in the cells isolated from all rats, including the heaviest (oldest). This finding shows that the potential of cells to develop into adipocytes also seems to exist in the adult rat under certain circumstances.