Microspheres (MS) have been proposed for use as oral vaccine delivery vehicles (VDV); however, due to poor and variable absorption their clinical utility is limited. The effects of size, zeta-potential, and surface hydrophobicity on the translocation (P-T) permeabilities of polystyrene (PS) MS with varying surface functional groups (amidine, carboxyl, carboxylate-modified [CML], and sulfate) were determined through gastrointestinal (GI) mucin. P-T were determined, under steady-state conditions, using a modified Ussing-type diffusion chamber and a mucin packet developed for use with the Transwell-Snapwell system. P-T followed the Stokes-Einstein relationship, demonstrating the limited ability of larger MS (>0.5 mu m) to diffuse through the mucin layer. P-T also varied according to the surface characteristics. Even though the zeta-potential did not correlate with the transport of MS through mucin, surface ionization appears to be important in MS translocation. The PS-amidine MS were significantly less hydrophobic and had a higher P-T than that of the other MS, suggesting that hydrophobicity is also a significant factor in MS transport through mucin. While these results suggest that mucin may be a significant barrier to the oral absorption of vaccines and VDVs in vivo, the rate-limiting barrier for the absorption of MS will be the intestinal mucosa.