This study extends our earlier work (T. Raclot and R. Groscolas. J. Lipid Res. 34: 1515-1526, 1993), which showed that, under norepinephrine-stimulated lipolysis, fatty acids of rat retroperitoneal fat cells are selectively mobilized. The present study examines whether this selective mobilization of fatty acids 1) is based on their proportions in adipose tissue, 2) is a metabolic feature common to all adipose tissues, and/or 3) depends on the lipolysis-stimulating agent. Rat fat cells with two markedly different fatty acid compositions were isolated from four white adipose tissues and treated with three lipolytic agents. Fatty acid composition of in vitro released free fatty acids was compared with that of fat cell triacylglycerols, the ratio of percent in free fatty acid to percent in triacylglycerol being defined as the relative mobilization rate (RMR). The RMR of individual fatty acids was related to their molecular structure. It increased exponentially with unsaturation for a given chain length and decreased with increasing chain length for a given unsaturation. The selectivity of fatty acid mobilization was similar regardless of the fatty acid composition of adipose tissue, the tissue location, and the lipolytic agent used. Under conditions of stimulated lipolysis, the selectivity of fatty acid mobilization is therefore a general metabolic feature of adipose tissue. Fatty acids with 16-20 carbon atoms and 4 or 5 double bonds had the highest RMR (from 1.4 to > 5), whereas fatty acids with 20-22 carbon atoms and 0 or 1 double bond had the lowest RMR (from 0.3 to 0.7). For the other fatty acids, RMR was close to unity.(ABSTRACT TRUNCATED AT 250 WORDS)