The R(-)- and S(+)-enantiomers of alpha-hydroxytolazoline, the benzylic hydroxy-substituted derivative of the alpha-adrenoceptor antagonist, tolazoline, were evaluated at alpha 1- and alpha 2-adrenoceptors in canine saphenous vein. Benzylic hydroxyl substitution of tolazoline in either the R(-) or S(+) configuration significantly decreased affinity at both alpha 1- and alpha 2-adrenoceptors. Differences in affinity between the R(-)- and S(+)-enantiomers were small, which is characteristic of imidazolines, but in marked contrast to phenethylamines where enantiomeric differences are large. The rank order of affinities at alpha 1- and alpha 2-adrenoceptors is tolazoline greater than S(+)-alpha-hydroxytolazoline = R(-)-alpha-hydroxytolazoline, which is different from that order predicted by the Easson-Stedman hypothesis (i.e., R(-) greater than S(+) = desoxy). The findings support our contention that phenethylamines and imidazolines interact differently with alpha-adrenoceptors.