Protein kinase D (PKD) exists as a family of structurally related enzymes that are activated through similar phosphorylation-dependent mechanisms involving protein kinase C (PKC). While individual PKD isoforms could in theory mediate distinct biological functions, previous studies identify a high level of functional redundancy for PKD1 and PKD2 in various cellular contexts. This study shows that PKD1 and PKD2 are activated in a stimulus-specific manner in neonatal cardiomyocytes. The α(1)-adrenergic receptor agonist norepinephrine selectively activates PKD1, thrombin and PDGF selectively activate PKD2, and endothelin-1 and PMA activate both PKD1 and PKD2. PKC activity is implicated in the α(1)-adrenergic receptor pathway that activates PKD1 and the thrombin- and PDGF-dependent pathways that activate PKD2. Endothelin-1 activates PKD via both rapid PKC-dependent and more sustained PKC-independent mechanisms. The functional consequences of PKD activation were assessed by tracking phosphorylation of CREB and cardiac troponin I (cTnI), two physiologically relevant PKD substrates in cardiomyocytes. We show that overexpression of an activated PKD1-S744E/S748E transgene increases CREB-Ser(133) and cTnI-Ser(23)/Ser(24) phosphorylation, but agonist-dependent pathways that activate native PKD1 or PKD2 selectively increase CREB-Ser(133) phosphorylation; there is no associated increase in cTnI-Ser(23)/Ser(24) phosphorylation. Gene silencing studies provide unanticipated evidence that PKD1 down-regulation leads to a compensatory increase in PKD2 activity and that down-regulation of PKD1 (alone or in combination with PKD2) leads to an increase in CREB-Ser(133) phosphorylation. Collectively, these studies identify distinct roles for native PKD1 and PKD2 enzymes in stress-dependent pathways that influence cardiac remodeling and the progression of heart failure.