A simple and sensitive enzymatic method for determination of plasma and serum fatty acids (FAs) is described. The method is based on acylation of long chain FAs by a bacterial acyl-CoA synthetase (ACS) producing equivalent amounts of acyl-CoA and AMP. AMP production was measured using the coupled reaction of myokinase (MK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) allowing fluorinate detection of NADH. Two moles of NAD were produced per mole of FA acylated. Concentrations of substrates and enzymes were kept as low as possible maintaining the ACS reaction as rate limiting. Addition of fat-free human serum albumin (HSA) to standards reduced initial reaction rates but did not affect end-point fluorescence levels. Triton X-100 partly counteracted the inhibition by HSA. To keep albumin concentration low, plasma or serum samples were diluted by 1:400. Duplicate measurements of plasma or serum FA concentrations between 0 and 2 mmol l-1 can then be performed on 5 microliters samples with intra- and inter-assay variation coefficients of 1.7 and 4% respectively.