All three subtypes of β‐adrenoceptors are coupled to stimulation of adenylyl cyclase activity via the stimulatory guanine‐nucleotide‐binding protein. Nevertheless, the β3 adrenoceptor (β3‐AR) differs significantly from the other subtypes in terms of pharmacology. Most strikingly, it recognizes as agonists several compounds acting as potent β1‐AR and β2‐AR antagonists. Furthermore, the human β3‐AR is quite different from the animal β3‐AR. Molecular modelling studies followed by site‐directed mutagenesis was used here to identify some of the amino acid residues which may be implicated in ligand binding and signal transduction of the β3‐AR. Three contiguous residues, valine‐leucine‐alanine, which are present in the first transmembrane domain at positions 48−50 of the human receptor but are absent in all known rodent sequences, were thought to be important for species specificity. When these three residues were deleted from the human receptor, no ’rodent‐like' pharmacological profile was obtained in terms of either binding or adenylyl cyclase activation. Glycine at position 53, also in the first transmembrane domain in the human β3‐AR, has been suggested to participate in β2‐/β3‐AR subtype selectivity. Replacement of this glycine residue by phenylalanine, which is the residue present at the homologous position in the human β2‐AR, left the β3‐AR pharmacological profile unaltered in terms of specificity and selectivity. Aspartate residue 117, in the third transmembrane domain, has been found to be essential for ligand binding and consequently adenylyl cyclase activation in several bioamine receptors. When this residue was replaced by a leucine residue in the β3‐AR, ligand binding and signal transduction were suppressed. Finally, replacement of asparagine at position 312 in the sixth transmembrane domain by an alanine residue, led to alterations in the signal‐transduction pathway.