Fatty acyl-CoA synthetase purified from rat liver microsomes was immobilized on either CNBr-activated Sepharose 4B or activated CH-Sepharose 4B, and the enzymatic activities of the syntheses of CoA esters from lignoceric acid (C24:0) and palmitic acid (C16:0) were studied and compared. The ratio of activities of the synthesis of lignoceroyl-CoA to palmitoyl-CoA increased 4.5 fold with CH-Sepharose, but only slightly with CNBr-Sepharose. The effects of a detergent and chaotropic agent on both substrates were significantly altered by the immobilization. The results of this study thus indicate that the stability and fatty acid specificity of fatty acyl-CoA synthetase are significantly affected by the physical state of the enzyme.