Gas chromatography is considered to be the reference method for ethyl alcohol determination. However, enzymatic ethanol assays have been developed for use in the clinical laboratory by several commercial vendors. Essentially, these assays utilize the oxidation of ethyl alcohol to acetaldehyde with concurrent reduction of nicotinamide adenine dinucleotide (NAD) to NADH while monitoring the increase in absorbance at 340 nm. The increase in absorbance is theoretically proportional to the ethanol concentration in the sample. Previously, several authors reported that increased concentrations of lactate and lactate dehydrogenase (LDH) can cause false-positive results with certain enzymatic ethyl alcohol assays. In the present investigation, we further studied the interference of lactate and LDH in three enzymatic assays. Apparent ethyl alcohol concentrations in serum spiked with lactate and LDH, as well as patient and autopsy samples, were determined by the Syva, Abbott, and Roche enzymatic assays and by gas chromatography. The effect of coenzyme depletion on the rate of reaction and the interference of hemolysis were also investigated. Based on our results we suggest that coenzyme depletion plays a major role in the severity of the false-positive ethyl alcohol result, and the interference from hemolysis has a negligible effect on these results. We also confirm the previous studies in showing that elevated serum-lactate and LDH concentrations can result in varying degrees of false-positive ethyl alcohol concentrations in the three enzymatic assays. This should be taken into consideration in the management of patients in a tertiary care medical center.