Immunological activation of T cells enhances synthesis of acetylcholine (ACh) and transcription of choline acetyltransferase (ChAT), M5 muscarinic ACh receptor (mAChR) and acetylcholinesterase (AChE). Stimulation of mAChRs on T and B cells causes oscillating Ca(2+)-signaling and up-regulation of c-fos expression; moreover, M1 mAChRs play a crucial role in the differentiation of CD8(+) T cells into cytolytic T lymphocytes. Collectively, these findings suggest that immune cell function is regulated by its own cholinergic system. Bearing that in mind, we tested whether immune function can be regulated via mAChR-mediated pathways by immunizing combined M1 and M5 mAChR knockout (M1/M5 KO) and wild-type (WT) C57BL/6JJcl mice with ovalbumin (OVA) and measuring serum IgG1 and IgM 1 wk later. We found that serum levels of total and anti-OVA-specific IgG1 were significantly lower in M1/M5 KO than WT mice, though there was no difference in serum levels of total and anti-OVA-specific IgM between the two genotypes. Secretion of interleukin (IL)-6 from activated spleen cells was significantly reduced in M1/M5 KO mice, whereas there was no significant change in gamma interferon secretion. Expression of AChE mRNA was significantly reduced in activated spleen cells from M1/M5 KO mice. These results suggest that M1 and/or M5 mAChRs are involved in regulating cytokine (e.g., IL-6) production, leading to modulation of antibody class switching from IgM to IgG1, but are not involved in the initial generation of the antibody response. They also support the notion that a non-neuronal cholinergic system is involved in regulating immune cell function.