—Recent studies have shown that chronic β-adrenergic receptor (β-AR) stimulation alters cardiac myocyte survival in a receptor subtype-specific manner. We examined the effect of selective β₁- and β₂-AR subtype stimulation on apoptosis induced by hypoxia or H₂O₂ in rat neonatal cardiac myocytes. Although neither β₁- nor β₂-AR stimulation had any significant effect on the basal level of apoptosis, selective β₂-AR stimulation protected myocytes from apoptosis. β₂-AR stimulation markedly increased mitogen-activated protein kinase/extracellular signal–regulated protein kinase (MAPK/ERK) activation as well as phosphatidylinositol-3′-kinase (PI-3K) activity and Akt/protein kinase B phosphorylation. β₁-AR stimulation also markedly increased MAPK/ERK activation but only minimally activated PI-3K and Akt. Pretreatment with pertussis toxin blocked β₂-AR–mediated protection from apoptosis as well as the β₂-AR–stimulated changes in MAPK/ERK, PI-3K, and Akt/protein kinase B. The selective PI-3K inhibitor, LY 294002, also blocked β₂-AR–mediated protection, whereas inhibition of MAPK/ERK activation at an inhibitor concentration that blocked agonist-induced activation but not the basal level of activation had no effect on β₂-AR–mediated protection. These findings demonstrate that β₂-ARs activate a PI-3K–dependent, pertussis toxin–sensitive signaling pathway in cardiac myocytes that is required for protection from apoptosis-inducing stimuli often associated with ischemic stress.