The mechanism of S-100 protein release from adipocytes, which is apparently coupled with lipolytic activity, was investigated in vitro using rat epididymal fat pads. The S-100 protein release was increased severalfold by 10 microM epinephrine in the medium containing a low concentration (less than 5 mg/ml) of albumin, but the release was enhanced only slightly when the medium contained a high concentration (more than 20 mg/ml) of albumin. On the other hand, the maximum rate of free fatty acid release measured simultaneously was observed in medium containing more than 20 mg/ml albumin. The rate of S-100 protein release was found to be closely related to the concentrations of both albumin added to the incubation medium and fatty acids released into it, and the rate was increased under conditions wherein the molar ratio of fatty acid/albumin was greater than 6. The S-100 protein release from fat pads was also enhanced solely by the addition of an excess amount (6 mM) of palmitic acid or oleic acid. The basal release of S-100 protein at a high concentration of albumin in the fat pads of diabetic or long-term starved rats, in which the fatty acid level in adipocytes is known to be enhanced, was about 7- and 2-fold higher, respectively, than that of control fed rats. These results suggest that S-100 protein in adipocytes is released under conditions in which the fatty acids being produced are not released promptly and are accumulated in the cells.