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The activation of docosahexaenoic acid by rat brain microsomes was studied using an assay method based on the extraction of unreacted [1-14C]docosahexanoic acid and the insolubility of [1-14C]docosahexaenoyl-CoA in heptane. This reaction showed a requirement for ATP, CoA, and MgCl2 and exhibited optimal activity at pH 8.0 in the presence of dithiothreitol and when incubated at 45 °C. The apparent Km values for ATP (185 µM), CoA (4.88 µM), MgCl2 (555 µM) and [1-14C]docosahexaenoic acid (26 µM) were determined. The presence of bovine serum albumin or Triton X-100 in the incubation medium caused a significant decrease in the Km and Vm values for [1-14C]docosahexaenoic acid. The enzyme was labile at 45 °C (t1/2 : 3.3 min) and 37 °C (t1/2 : 26..5 min) and lost 36% of its activity after freezing and thawing. The transition temperature (Tc) obtained from Arrhenius plot was 27 °C with the activation energies of 74 kJ/mol between 0 °C and 27 °C and 30 kJ/mol between 27 °C and 45 °C. [1-14C]Palmitic acid activation in rat brain and liver microsomes showed apparent Km values of 25 µM and 29 µM respectively, with V values of 13 and 46 nmol x min-1. The presence of Triton X-100 (0.05%) in the incubation medium enhanced the V value of the liver enzyme fourfold without affecting the Km value. Brain palmitoyl-CoA synthetase, on the other hand, showed a decreased Km value in the presence of Triton X-100 with unchanged V. The Tc obtained were 25 °C and 28 °C for brain and liver enzyme with an apparent activation energy of 109 and 24 kJ/mol below and above Tc for brain enzyme and 86 and 3.3 kJ/mol for liver enzyme. The similar results obtained for the activation of docosahexaenoate and palmitate in brain microsomes suggest the possible existence of a single long-chain acyl-CoA synthetase. The differences observed in the activation of palmitate between brain and liver microsomes may be due to organ differences. Fatty acid competition studies showed a greater inhibition of labeled docosahexaenoic and palmitic acid activation in the presence of unlabeled unsaturated fatty acids. The Ki values for unlabeled docosahexaenoate and arachidonate were 38 µM and 19 µM respectively for the activation of [1-14C]docosahexaenoate. In contrast, the activation of unlabeled saturated fatty acids for activation of labeled docosahexaenoate is much less than that for activation of labeled palmitate. The preferential inhibition by unsaturated fatty acids, especially the polyunsaturated fatty acids, of the activation of docosahexaenoic and palmitic acids, as compared to inhibition by saturated fatty acids may be the results of the higher affinity or ready accessibility of these more unsaturated substrates to the membrane-bound enzyme. [ABSTRACT FROM AUTHOR]