The permeation selectivity of a series of Langmuir-Blodgett (LB) films derived from calix[n]arene-based amphiphiles of varying diameters (i.e., 2a, 2b, and 2c) has been examined using poly[1-(trimethylsilyl)-1-propyne] (PTMSP) as support material and He and SF6 as the permeants. Examination of He/SF6 flux ratios as a function of calix[n]arene size, number of LB layers, transfer speed, and orientation of the substrate relative to the direction of monolayer flow, indicate that (i) permeation across these LB films is governed by diffusion between neighboring molecules and not through the molecular pores of the calix[n]arenes and (ii) the mechanics of LB film fabrication can have a significant influence on defect formation.