A method comparison study for the determination and quantitation of nonesterified fatty acids (NEFAs) in serum, using the commercial "NEFA C" enzymatic test kit, was performed using the spectrophotometric method recommended by the manufacturer and a modified procedure optimized for the use of a microplate reader, a 96-well microtiter plate, and small sample volumes (10 microl). Linearity, sensitivity, and precision using the test kit were determined for each method of detection. The assay was linear from 0 to 1.97 mEq/liter for both procedures, and the limits of detection were determined to be 0.22 (+/- 0.074) and 0.05 (+/- 0.002) mEq/liter for the spectrophotometer and microplate reader, respectively. Pairs of measurements for bovine serum samples were compared and evaluated by a mean difference plot method and not regression analysis, a method that has been shown to be inappropriate for method comparison studies. The difference plot was used to evaluate the systematic bias between the 2 methods. Random error is reported on the basis of SD differences, and "limits of agreement" are used to describe the maximum differences likely to occur between the 2 methods. Results suggest that the microplate reader method can be used reliably in place of the recommended spectrophotometric method. The microplate reader method is preferred because of its high throughput capabilities, simultaneous analysis of all the standards and samples, use of small sample and reagent volumes, and reduction in labor requirements and costs.