Site-directed mutagenesis and photoaffinity labeling experiments suggest the existence of at least two distinct binding orientations for aryloxypropanolamine competitive antagonists in the beta-adrenergic receptor (beta-AR), one where the aryloxy moiety is located near transmembrane alpha-helix 7 (tm 7) and another where it is near tm 5. To explore a hydrophobic pocket involving tms 1, 2, 3, and 7 for potential aryloxy interaction sites, we selected Tyr(356(7.43)) and Trp(134(3.28)) in the rat beta(1)-AR for site-directed mutagenesis studies. Ser(190(4.57)) was also investigated, as the equivalent residues are known antagonist interaction sites in the muscarinic M(1) and the dopamine D(2) receptors. Binding affinities (pK(i)) of a series of structurally diverse aryloxypropanolamine competitive antagonists were determined for wild type and Y356A, Y356F, W134A, and S190A mutant rat beta(1)-ARs stably expressed in Chinese hamster ovary cells. To visualize possible antagonist/receptor interactions, the compounds were docked into a three-dimensional model of the wild-type rat beta(1)-AR. The results indicate that Tyr(356(7.43)) is an important aromatic interaction site for five of the eight competitive antagonists studied, whereas none of the compounds appeared to interact directly with Trp(134(3.28)). Only two of the competitive antagonists interacted with Ser(190(4.57)) on tm 4. Overall, the results extend our understanding of how beta(1)-AR competitive antagonists bind to the hydrophobic pocket involving tms 1, 2, 3, and 7; highlight the importance of Tyr(356(7.43)) in this binding pocket; and demonstrate the involvement of tm 4 in competitive antagonist binding.