It has previously been shown that oral or intravenous ethanol lowers the serum folate level as determined by theLactobacillus caseiassay. To exclude an assay artifact, similar changes were sought with a new radioassay for serum folate. Serial serum folate levels were determined by both theL. caseiassay and the radioassay in three normal volunteers during and following short-term ingestion of ethanol, and the results were compared to control regimens during which no ethanol was ingested. In all subjects the serum folate level fell during ethanol ingestion and rose after ethanol intake was stopped. The degree of the ethanol-induced fall in serum folate level seemed to correlate with the amount of ethanol consumed. The results of the radioassay essentially paralleled those of theL. caseiassay, indicating that the fall in serum folate level induced by ethanol is not an artifact of the microbiological assay. Although alternate mechanisms remain to be excluded, it is possible that ethanol interferes with the formation of and/or the release of N-5-methyltetrahydrofolic acid, the main folate metabolite in the serum of fasting subjects.