The first putative extracellular domains of both m1 and m2 muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y), and threonine (T). This triplet is repeated as LYTLYT in m2 receptors. However, it is repeated in a transposed fashion (LYTTYL) in the sequence of m1 receptors. In this work we employed site-directed mutagenesis to investigate the possible significance of this unique sequence diversity in determining the distinct differential drug-receptor interaction at the two receptor subtypes. Mutation of the LYTTYL sequence of m1 receptors to the corresponding m2 receptor LYTLYT sequence, however, did not significantly change the binding affinity of the agonist carbachol or the affinity of the majority of a series of receptor antagonists which are able to discriminate between wild-type m1 and m2 receptors. The reverse mutation at the m2 receptor also did not modify agonist affinity, but altered affinity of several receptor subtype-selective antagonists. The magnitude of affinity changes, however, was small, and the direction of these changes was opposite to what would be expected if the m2 receptor LYTLYT seqence were important for determining the binding profile of m2-receptor-selective antagonists. Our data suggest that the LYTTYL-LYTLYT sequence differences between ml and m2 muscarinic receptors are not important for determining receptor pharmacology.