Both M₁ and M₂ muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T) at C-terminus ends of the second putative transmembrane domains. This triplet is repeated as LYT-LYT in M₂ receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-TYL) in the sequence of M₁ receptors. In this work, we employed site-directed mutagenesis to investigate the possible significance of this unique sequence diversity for determining the distinct differential cellular function at the two receptor subtypes. Mutation of the LYTTYL sequence of M₁ receptors to the corresponding M₂ receptor LYTLYT sequence did not result in a significant change in the binding affinity of the agonist carbachol. The reverse mutation at the M₂ receptor also did not modify agonist affinity. Surprisingly, the LYTLYT M₁ receptor mutant demonstrated markedly enhanced coupling to activation of phospholipase C without a change in its coupling to increased cyclic AMP formation. There was also an enhanced receptor sensitivity in transducing elevation of intracellular Ca²⁺. On the other hand, the reverse LYTLYT{\rightarrow}LYTTYL mutation in the M₂ receptor did not alter its coupling to inhibition of adenylate cyclase, but slightly enhanced its coupling to stimulation of phosphoinositide (PI) hydrolysis. Our data suggest that the LYTTYL/LYTLYT sequence differences between M₁ and M₂ muscarinic receptors are not important for specifying ligand binding and coupling of various subtypes of muscarinic receptors to different cellular signaling pathways although they might play a role in the modulation of muscarinic reseptor coupling to PI hydrolysis.