Previous studies have suggested that protein kinase C (PKC) is involved in heat shock protein (Hsp)–mediated cardioprotection. Therefore, we wanted to determine whether overexpression of Hsps modulates PKC expression, which will give us further insight into understanding the mechanism by which Hsps and PKC interact to protect cells from stress-induced injury. Specifically, we overexpressed the inducible form of Hsp70 (Hsp70i) or Hsp90 in rat neonatal cardiomyocytes and evaluated PKCδ or PKCϵ expression by immunoblotting and immunofluorescent confocal microscopy. Western analysis showed that overexpression of Hsp70i or Hsp90 decreased PKCϵ expression. However, overexpression of Hsp70i or Hsp90 did not modify PKCδ expression over control levels. Overexpression of constitutively active PKCδ or PKCϵ increased Hsp70i expression over control levels. The data suggest that overexpression of Hsps differentially modulates expression of PKC isoforms in rat neonatal cardiomyocytes. Furthermore, PKC may directly play a role in Hsp-mediated cardioprotection by upregulating Hsp70i expression.