The reduction of parasympathetic nerve activity by the treatment with ethylcholine mustard aziridinium ion (AF64A) in vivo induced both specific and non-specific supersensitivities in the rat iris sphincter (Tanaka et al., 1999). Changes in the expression of muscarinic receptor subtypes, which could be a cause of specific supersensitivity induced by the treatment with AF64A, were examined using competitive PCR techniques. Muscarinic receptor population is composed of m2, m3, and m4 subtypes in the rat iris (Furuta et al., 1998). Interestingly, m4 mRNA was much more abundantly expressed than m2 and m3 in the rat iris sphincter. The treatment with AF64A significantly increased the mRNA levels of m2 and m3 subtypes to 370 and 330% of the control but not that of m4 (approximately 90% of the control). In addition, the total protein contents were increased to approximately 125% of the control. The up-regulation of the mRNA levels of m2 and m3 subtypes by the treatment with AF64A was significant when they were compensated for the increase in total protein contents. The down regulation of m4 mRNA expression was not significant even after being corrected for the protein content. These results suggest that the up-regulation of the mRNA levels of m2 and m3 subtypes may be, at least in part, responsible for the supersensitivity to muscarinic agonists after the treatment with AF64A in vivo.