The protein kinase C (PKC) family consists of serine/threonine protein kinases that play important roles in signal transduction, cell proliferation, and tumor formation. Recent studies found that PKCs are commonly overexpressed in human tumors, including soft tissue sarcoma (STS). Overexpression of PKCs contributes to invasion and migration of tumor cells and induction of angiogenesis. PKC can also phosphorylate the multidrug resistance (MDR) gene-encoded P-glycoprotein and induce MDR phenotype. Our previous studies showed that mutation of p53 enhanced STS metastasis and mediated the MDR phenotype. Restoring wild type (WT) p53 in STS cells containing mutant p53 sensitized the cells to chemotherapy. In the present study, we found that PKCalpha protein expression is inhibited by WT p53 partly due to reduced PKCalpha mRNA expression in STS cells, but p53 does not affect PKCalpha mRNA stability. Deletion and mutation analysis of the PKCalpha promoter fused to the luciferase reporter gene identified a Sp1 binding site (-244/-234) in the PKCalpha promoter that is required for p53-mediated inhibition of PKCalpha promoter activity. More importantly, PKCalpha phosphorylates and activates MDR1 P-glycoprotein, whereas inhibition of PKCalpha by p53 leads to decreased MDR1 phosphorylation in STS cells, which sensitizes STS cells to chemotherapeutic agents. These data indicate that WT p53 may resensitize STS to chemotherapeutic agents by reducing MDR1 phosphorylation via transcriptional repression of PKCalpha expression. Thus, molecular-based therapies targeting mutant p53 and PKCalpha may be an effective new strategy to improve chemotherapeutic efficacy in STS.