Technical Abstract: Fumonisins (FB) are inhibitors of ceramide synthase (CS), a key enzyme in the sphingolipid biosynthetic pathway. Nixtamalization, the alkali processing of corn, reduces the total FB in corn products. However, it is unclear whether hidden or more toxic FB metabolites are formed. To address this issue, the biological activity of FB contaminated corn before and after nixtamalization was determined in vitro. Sphinganine elevation was used as a biomarker for FB inhibition of CS in LLC-PK1 and Vero cells. However, clean corn extracts, but not extracts of nixtamalized products, were toxic to cells. The objectives of this study were 1) determine a non-toxic exposure for corn extracts and 2) determine if a reduction in total detectable fumonisins by nixtamalization results in a similar reduction in biological activity. LLC-PK1 cells were treated for various times with various amounts of residues of corn extracts dissolved in growth medium. There was a dose and time dependent decrease in ATP-dependent dome formation and tight junction integrity based on visual observation. A 6h exposure to the equivalent of 0.086 g corn/ml of growth medium was used for subsequent experiments. In Vero cells exposure was for 48 h at 0.04 g corn equivalents/ml growth medium. Ceramide synthase inhibition by extracts of contaminated corn and nixtamalized products was determined. There was a significant decrease in sphinganine elevation in cells exposed to nixtamalized products compared to cells exposed to the FB contaminated corn used to make the products. These results indicate that nixtamalization-of FB-contaminated corn does not produce increased fumonisin-like biological activity; results consistent with the conclusion that hidden or more toxic FB metabolites are not formed during the nixtamalization process.