The nature of the immune response following ocular immunization with a protein and a polysaccharide and the effects such immunization have on the activities of cholinergic enzymes in the lacrimal glands of BALB/c mice were examined. Lacrimal glands are highly innervated by sympathetic and parasympathetic nerve fibres and are involved in mucosal immunity and therefore are excellent sites to study neuro-immune interactions. In this report, a T-lymphocyte-dependent protein antigen, keyhole limpet haemocyanin (KLH) and a T-lymphocyte-independent polysaccharide antigen, dextran (DEX) were administered topically to the eyes or intraperitoneally injected. Both routes of immunization produced a strong serum antibody response when KLH was the antigen. DEX, however, evoked a serum antibody response only after intraperitoneal administration. Eosin-haematoxylin staining indicated no histological abnormality or inflammatory changes in any immunized lacrimal glands, but immuno-staining revealed that only in the KLH-treated tissues were IgG-producing plasma cells discernible. Furthermore, KLH-specific antibody was also detectable using an immuno-blot assay in lacrimal glands. Polymerase chain reaction analysis with cytokine-specific primers revealed induction of interleukin-4 (lL-4) in KLH-treated lacrimal glands, but not in DEX or unimmunized tissues. Thus, the nature of the antigen seems important in the induction of the immune response in lacrimal glands. To delineate the effects that immunogenic differences might have on the activities of the cholinergic enzymes, choline acetyl-transferase (ChAT) and acetylcholinesterase (AChE) were assayed using radiolabelled substrates and measuring labelled products. Both ChAT and AChE activities were influenced following KLH immunization, while DEX had only transient effects on ChAT. This is possibly due to the fact that KLH, a protein antigen, is the effective inducer of the specific immune response in the lacrimal gland, while DEX is not. Experimental Physiology (2001) 86.2, 169-176.