Two types of acyl-CoA synthetase (ACS), designated ACS1 and ACS2, are structurally similar isozymes with different tissue distributions. The two enzymes are organized into the following five regions: an NH2 terminus; two luciferase-like regions; a linker connecting the luciferase-like regions; a COOH terminus. Under the control of a lac promoter, rat ACS1 and ACS2 were overproduced in Escherichia coli and purified to homogeneity. The specific activities of the purified ACS1 and ACS2 were 26.2 mumol.min-1.mg-1 and 7.4 mumol.min-1.mg-1, respectively, and the most efficiently utilized saturated fatty acids were those with 10-18 carbon atoms. Among unsaturated fatty acids with 16-22 carbon atoms, the most preferred substrates were palmitoleate, oleate and linoleate for ACS1, and, for ACS2, oleate, arachidonate, eicosapentaenoate and docosahexaenoate. To determine the functionally important regions in the ACS isozymes, we constructed five ACS1 mutants lacking each of the five regions. Introduction of these mutants into E. coli revealed that all five regions in ACS1 are required for functional expression of the enzyme in E. coli; deletion of any one of the five regions almost completely abolished the enzyme activity.