Myocardial β -adrenergic receptors (β -ARs) consist ofβ₁ - and β₂-subtypes, which mediate distinct signaling mechanisms. We examined whichβ -AR subtype mediates cardiac hypertrophy. The β₂-subtype is predominant in neonatal rat cardiac myocytes (β₁, 36%vβ₂, 64%), while theβ₁ -subtype predominates in the adult rat heart (59%v 41%). Stimulation of cultured cardiac myocytes in vitro with isoproterenol (ISO), an agonist for β₁- andβ₂ -ARs, caused hypertrophy of myocytes along with increases in transcription of atrial natriuretic factor (ANF) and actin reorganization. All of these ISO-mediated myocyte responses in vitro were inhibited by aβ₁ -AR antagonist, betaxolol, but not by a β₂-AR antagonist, ICI 118551. Pertussis toxin failed to affect ISO-induced increases in total protein/DNA content and ANF transcription in vitro. ISO increased LV weight/body weight and ANF transcription in the adult rat in vivo, which were also inhibited by betaxolol but not by ICI 118551. These results suggest that β -AR stimulated hypertrophy is mediated by theβ₁ -subtype and by a pertussis toxin-insensitive mechanism