The cAMP-dependent protein kinase types I (PKA-I) and II (PKA-II), composed of identical catalytic (C) subunits but distinct regulatory (R) subunits (RI versus RII), are expressed in a balance of cell growth and differentiation. Distortion of this balance may underlie tumorigenesis and tumor growth. Here, we used PC3M prostate carcinoma cells as a model to overexpress wild type and mutant R and C subunit genes and examined the effects of differential expression of these genes on tumor growth. Only the RIIbeta and mutant RIalpha-P (a functional mimic of RIIbeta) transfectants exhibited growth inhibition in vitro, reverted phenotype, and apoptosis, and inhibited in vivo tumor growth. DNA microarrays demonstrated that RIIbeta and RIalpha-P overexpression upregulated a cluster of differentiation genes, while downregulating transformation and proliferation signatures. Overexpression of RIalpha and Calpha, which upregulated PKA-I, elicited the expression signatures opposite that elicited by RIIbeta overexpression. Total colocalization of Calpha and RIIbeta seen by confocal microscopy in the RIIbeta cell nucleus supports the opposed genomic regulation demonstrated between Calpha and RIIbeta cells. Differential expression of PKA R subunits may therefore serve as a tumor-target-based gene therapy for PC3M prostate and other cancers.