Mice were intranasally administered enterotoxigenic Escherichia coli colonization factor CS6 encapsulated in poly(DL-lactide-co-glycolide) microspheres (CS6-PLG), with immune response measured and compared to that of similarly administered native CS6 and CS6 plus mutant heat-labile enterotoxin mucosal adjuvant (CS6+mLT). Native CS6 and the CS6-PLG microspheres administered intranasally to mice induced serum IgG responses, with the CS6-PLG microspheres inducing a significantly greater (P<0.001) response than native CS6. Following intranasal administration of native CS6, no fecal IgG and IgA responses were measured; however, the CS6-PLG microspheres induced significantly greater (P<0.001) fecal IgG and IgA responses than native CS6. The coadministration of the mLT mucosal adjuvant with CS6 induced significantly greater serum (P<0.001) and fecal (P<0.01) responses than the CS6-PLG microspheres. However, following intranasal administration of the mLT adjuvant, the mice showed definite signs of distress, indicating an adverse reaction to the mLT. Thus, this brings into question the safety of the mLT and its use as an intranasal adjuvant. In contrast, the PLG-microspheres administered intranasally caused no noticeable distress to the mice. The results obtained in this study indicate that the encapsulation of CS6 in PLG-microspheres administered intranasally to mice acted in an adjuvant capacity to enhance the CS6 immune response.