Site-specific and sex differences in fatty acid/triacylglycerol substrate cycling in adipose tissue and muscle were found in vivo in adult hamsters (Phodopus sungorus) fed ad libitum, both at rest and immediately after moderate exercise. In adipose tissue, rates of fatty acid/triacylglycerol cycling at rest were highest in two small intermuscular depots and lowest in the groin, behind forelimb, epididymal and kidney sites. Cycling rates were significantly higher in the groin and in the two depots around the forelimb in females. Following an hour of exercise in a hamster ball, the rates of fatty acid/triacylglycerol cycling rose significantly in the intermuscular and certain superficial adipose fatty depots of both sexes, but the increases were more uniform in males. In females only, cycling rates in the skeletal muscles also increased significantly. The rates of fatty acid/triacylglycerol cycling in adipose tissue correlate closely with the site-specific activities of hexokinase and phosphofructokinase, but not with mean adipocyte volume per se. The data are consistent with the hypotheses that adipose depots are depleted selectively during exercise and that there are sex differences in the pattern of lipid mobilization.