The enantiomers of formoterol (R;R and S;S) and their diastereomers (R;S and S;R) were synthesized and purified using a new procedure which required the preparation of the (R;R)- and (S;S)-forms of N-(1-phenylethyl)-N-(1-(ρ-methoxyphenyl)-2-propyl)-amine as important intermediates. The enantiomeric purity obtained was greater than 99.3%, usually > 99.7%. The four stereoisomers were examined with respect to their ability to interact in vitro with β-adrenoceptors in tissues isolated from guinea pig. The effects measured were (1) relaxation of the tracheal smooth muscle (mostly β2), (2) depression of subtetanic contractions of the soleus muscle (β2), and (3) increase in the force of the papillary muscle of the left ventricle of the heart (β1). All enantiomers caused a concentration-dependent and complete relaxation of the tracheal smooth muscle which was inhibited by propranolol. The order of potency was (R;R) > > (R;S) = (S;R) > (S;S). There was a 1,000-fold difference in potency between the most and the least potent isomer. The presence of the (S;S)-isomer did not affect the activity of the (R;R)-isomer on the tracheal smooth muscle. Also on the skeletal and cardiac muscles (R;R)-formoterol was more potent than its (R;S)-isomer. The selectivity for β2-adrenoceptors appeared to be slightly higher for the (R;R)-isomer than for the (R;S)-isomer. The potency of the (S;R)- and (S;S)-isomers on the papillary muscle was too low to be determined accurately. The present study shows that determination of enantiomeric ratios and conclusions regarding structure–effect relationships are critically dependent on a very high degree of stereochemical purity.