Elevated protein kinase C β II (PKCβ II) expression develops during heart failure and yet the role of this isoform in modulating contractile function remains controversial. The present study examines the impact of agonist-induced PKCβ II activation on contractile function in adult cardiac myocytes. Diminished contractile function develops in response to low dose phenylephrine (PHE, 100nM) in controls, while function is preserved in response to PHE in PKCβ II-expressing myocytes. PHE also caused PKCβ II translocation and a punctate distribution pattern in myocytes expressing this isoform. The preserved contractile function and translocation responses to PHE are blocked by the inhibitor, LY379196 (30nM) in PKCβ II-expressing myocytes. Further analysis showed downstream protein kinase D (PKD) phosphorylation and phosphatase activation are associated with the LY379196-sensitive contractile response. PHE also triggered a complex pattern of end-target phosphorylation in PKCβ II-expressing myocytes. These patterns are consistent with bifurcated activation of downstream signaling activity by PKCβ II.