The binding to bovine neurophysin of lysine-vasopressin and of lysine-vasopressin selectively deuterated at the protons ortho to the tyrosine hydroxyl was studied by proton n.m.r. and equilibrium dialysis. The principal object of these studies was to investigate reports that, at standard salt concentrations, neurophysin contained a second site specific for vasopressin. At pH 6, the effects of neurophysin-I on the line-width, longitudinal relaxation rate and nuclear Overhauser properties of the lysine-vasopressin tyrosine ring protons were interpretable in terms of a slow-exchange 1:1 interaction between lysine-vasopressin and neurophysin. Additionally, n.m.r. competition studies between lysine-vasopressin and L-phenylalanyl-L tyrosinamide suggested 1:1 competition for a single binding site on neurophysin. No evidence pointing to a significant second lysine-vasopressin-binding site was obtained from the n.m.r. studies. The lack of a moderately strong second binding site for lysine-vasopressin at neutral pH was also indicated by equilibrium dialysis studies at relatively high free hormone concentrations. These studies demonstrated only a single thermodynamically significant site for either oxytocin or vasopressin and failed to confirm a reported effect of LiCl on the number of sites available to oxytocin. It is suggested that secondary sites for the hormones are probably markedly weaker and less specific than reported elsewhere.