Acetylcholine responses of the α1 subunit containing wild type mouse muscle nicotinic acetylcholine receptors (AChRs) expressed in Xenopus oocytes are readily blocked by α-Bungarotoxin (α-Bgt, Boulter et al., 1987). Some studies suggest that α1 Proline 194 provides a structure necessary for strong binding of toxin (Kachalsky et al., 1995), whereas other studies contradict this (Tomaselli et al., 1991).To resolve whether proline really is essential, we have investigated the properties of a muscle type receptor containing α1 with Serine in place of Proline at position 194. Control acetylcholine (100nM) responses were recorded in normal ringer before incubation with α-Bgt (100 nM) for 18 minutes (mins). After reperfusion with control ringer, recovery responses were measured at 2, 10, 20, 30, 40 and 50 mins. Recovery (% of the initial control, ± S.E.M.) after 50 mins of washing was 8.04 ± 1.43%, n = 4 for α1(Ser 194) β1γδ receptors. Different oocytes also expressing α1(Ser 194) β1γδ receptors were then incubated with various α-Bgt concentrations for 17 min. The estimated IC₅₀ values for α1(Ser 194) β1γδ was 1.1 X 10^-8M (n = 5-10, at each toxin concentration 1-100 nM). We also used the method of Jenkinson, D.H., (1996) to obtain separate estimates of the microscopic association (k₊₁) and dissociation (k_-1) rate constants. The kvalue of toxin binding to α1(Ser 194) β1γδ receptors was 0.0020 nM^-1 min^-1,while the corresponding kvalue was 0.032 min^-1.From these rate constants, the equilibrium dissociation rate constant (K_D) for α-Bgt binding was calculated to be 15.65 nM. Taken together, these data suggest that the mouse muscle α1 subunit does not require the structural motif provided by Proline 194 in order to bind α-Bgt strongly. Therefore although determinants for toxin binding do occur in the 185-196 region of α1, Proline is not one of them. In order to harvest the oocytes, Xenopus were killed humanely in accordance with Human Office regulations.