In the search for new hypotensive agents a series of pyrrolidin-2-one derivatives was obtained with α-adrenoceptor blocking properties. The aim of the present study was to examine the possible involvement of other mechanisms in the observed hypotensive properties. In the present study the affinities for β1-adrenoreceptors, vasorelaxant effect and the involvement in NO pathway of pyrrolidin-2-one derivatives were assessed. In the next step compounds were also evaluated for their α1-adrenoreceptor subtypes in functional bioassays. The data from our experiments indicate that the hypotensive activity of tested pyrrolidin-2-one derivatives is a result of their α-adrenoceptor blocking properties and the compounds have stronger antagonist potency for the α1D- than for α1B-subtype. Among investigated compounds EP-46 is the most potent and selective antagonist for the α1D- and α1A- than for α1B-subtype. Compound EP-43 can enhance NO production additionally.