1. 1. Homogenates of rat epididymal fat pad, heart, kidney, lactating mammary gland, liver, skeletal muscle and small intestinal mucosa have been partitioned into a particulate and supernatant fraction. With reliable marker enzymes for the mitochondrial matrix and the cytosol: propionyl-CoA carboxylase and pyruvate kinase, the distributions of the acyl-CoA synthetase activities measured at 1 and 10 mM C 2, C 3 and C 4 over mitochondria and cytosol have been calculated. From these values an estimate was made of the k 0.5 of the fatty acids. 2. 2. A distinct fatty acid-activating enzyme was assumed to be present in one of the compartments when that fatty acid was activated with a K 0.5⩽ 1.5 mM in an amount of >13% of the total cellular activity. Adipose tissue, gut, liver and mammary gland, all organs of a high lipogenetic capacity, contained a cytosolic acetyl-CoA synthetase. At 1 mM acetate 60, 31, 77 and 83% of the total cellular activities in these organs were cytosolic in nature, with activities of 0.021, 0.32, 0.37 and 1.16 μmol C 2 activated per min per g wet weight, respectively. 3. 3. Mitochondrial acetyl-CoA and butyryl-CoA synthetases were found in adipose tissue, gut, heart, kidney, mammary gland and muscle. They were absent in liver. Adipose tissue and liver contained a mitochondrial propionyl-CoA synthetase with activities at 1 mM C 3 of 0.014 and 1.50 μmol C 3 activated per min per g wet weight, respectively. 4. 4. At 1 mM, C 2 was activated with decreasing rates by kidney, heart, mammary gland and gut (7.6-1.0 μmol C 2 activated per min per g wet weight). C 3 (1 mM) activation was about equal (1.6–1.9 μmol C 3 activated per min per g wet weight) in liver, kidney and heart. C 4 (1 mM) was activated with decreasing rates by heart, liver, kidney and gut (4.0-0.5 μmol C 4 activated per min per g wet weight) in the order given. 5. 5. The influence of the isolation method and the diet on fatty acid activation in small intestinal mucosal scrapings have been studied. To demonstrate the existence of cytosolic acetyl-CoA synthetase in fed animals a pre-treatment of everted intestine by low amplitude vibration has been found essential. Also C 16 activation was highly (95%) decreased in a non-pre-vibrated preparation. 24 h starvation lowered cytosolic C 2 and total C 16 activation by 90 and 80%, respectively. Refeeding of starved rats with a balanced fat-free diet, and not with sucrose only, gave the same cytosolic C 2 and total C 16 activation as normally fed rats. 6. 6. In guinea-pig heart, kidney, liver and muscle about the same partitions have been found as in the respective rat organs. The acetate activation in liver was a factor 6 lower. Acetate and butyrate activation in guinea-pig muscle was much higher (6 and 37 times, respectively).