We synthesized PYY-(1-36) (nonselective between Y1 and Y2 receptor subtype agonists), [Pro34]PYY (selective for Y1), and PYY-(3-36) (selective for Y2) to determine whether solution conformation plays a role in receptor subtype selectivity. The three peptides exhibited the expected specificities in displacing labeled PYY-(1-36) from cells transfected with Y1 receptors (dissociation constants = 0.42, 0.21, and 1,050 nM, respectively) and from cells transfected with Y2 receptors (dissociation constants = 0.03, 710, and 0.11 nM, respectively) for PYY-(1-36), [Pro34]PYY, and PYY-(3-36). Sedimentation equilibrium analyses revealed that the three PYY analogs were 80-90% monomer at the concentrations used for the subsequent circular dichroism (CD) and 1H-nuclear magnetic resonance (NMR) studies. CD analysis measured helicities for PYY-(1-36), [Pro34]PYY, and PYY-(3-36) of 42%, 31%, and 24%, suggesting distinct differences in secondary structure. The backbone 1H-NMR resonances of the three peptides further substantiated marked conformational differences. These patterns support the hypothesis that Y1 and Y2 receptor subtype binding affinities depend on the secondary and tertiary solution state structures of PYY and its analogs.