We report the first direct measurement of delta-6 desaturase and delta-9 desaturase (EC 1.3.99.3, acyl-CoA dehydrogenase) activities in the rat kidney. Crude renal cortical homogenates from alloxan-diabetic and from normal rats were assayed for delta-6 and delta-9 desaturase activities. The delta-6 desaturation pathway activity measured with 9,12-octadecadienoic acid (linoleic acid) as substrate was increased, while the delta-9 desaturation pathway measured with hexadecanoic acid (palmitic acid) as substrate was unchanged in diabetic renal cortex, suggesting that the two enzymes are regulated independently in this tissue. In contrast to the kidney, delta-6 desaturase pathway activity was unchanged and the delta-9 desaturase pathway activity was greatly depressed in diabetic liver. When exogenous long-chain acyl-CoA synthetase (EC 6.2.1.3; acid: CoA ligase, AMP-forming) was added to the delta-6 desaturase assay system, the rate of delta-6 desaturation in normal kidney increased to a rate similar to that found in diabetic kidney; rates in diabetic extracts were unchanged. These results suggest that the rate of fatty acid substrate activation to the coenzyme A ester limits the rate of delta-6 desaturation in normal renal cortex. These results also suggest that the rate of fatty acid activation by long-chain acyl-CoA synthetase activity is increased in diabetic renal cortex. Direct measurement of the activity of long-chain acyl-CoA synthetase demonstrated that its activity was indeed increased significantly in the renal cortex of diabetic rats.