A majority (84%) of G protein-coupled receptors have a proline (P5.50) in the middle of the fifth transmembrane domain. However, one of the unique structural features of cannabinoid receptors is the replacement of the conserved P5.50 by a leucine (L5.50). It has been shown that a conserved tyrosine (Y5.58), located at the cytoplasmic side of P5.50, is crucial for the signal transduction of several G protein-coupled receptors. We proposed that the replacement of P5.50 by L5.50 and the presence of the conserved Y5.58 in this context are important for the function of CB2. Mutating L5.50 to a proline abolished ligand binding, whereas mutating Y5.58 to an alanine resulted in a rightward shift of the competition binding curves. Both of these mutations led to a complete loss of the ability of cannabinoid agonists to inhibit forskolin-stimulated cAMP accumulation.