Ethanol may cause fetal alcohol spectrum disorders (FASD) in part by inhibiting cell adhesion mediated by the L1 neural cell adhesion molecule. Azialcohols photolabel Glu-33 and Tyr-418, two residues that are predicted by homology modeling to lie within 2.8 Å of each other at the interface between the Ig1 and Ig4 domains of L1 (Arevalo, E., Shanmugasundararaj, S., Wilkemeyer, M. F., Dou, X., Chen, S., Charness, M. E., and Miller, K. W. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 371-375). Using transient transfection of NIH/3T3 cells with wild type (WT-L1) and mutated L1, we found that cysteine substitution of both residues (E33C/Y418C-L1) significantly increased L1 adhesion above levels observed for WT-L1 or the single cysteine substitutions E33C-L1 or Y418C-L1. The reducing agent β-mercaptoethanol (βME) reversibly decreased the adhesion of E33C/Y418C-L1, but had no effect on WT-L1, E33C-L1, or Y418C-L1. Thus, disulfide bond formation occurs between Cys-33 and Cys-418, confirming both the close proximity of these residues and the importance of Ig1-Ig4 interactions in L1 adhesion. Maximal ethanol inhibition of cell adhesion was significantly lower in cells expressing E33C/Y418C-L1 than in those expressing WT-L1, E33C-L1, or Y418C-L1. Moreover, the effects of βME and ethanol on E33C/Y418C-L1 adhesion were non-additive. The cutoff for alcohol inhibition of WT-L1 adhesion was between 1-butanol and 1-pentanol. Increasing the size of the alcohol binding pocket by mutating Glu-33 to Ala-33, increased the alcohol cutoff from 1-butanol to 1-decanol. These findings support the hypothesis that alcohol binding within a pocket bordered by Glu-33 and Tyr-418 inhibits L1 adhesion by disrupting the Ig1-Ig4 interaction.