Protein kinase C (PKC) promotes cell survival in response to ionizing radiation in a variety of experimental models including human carcinoma, human glioblastoma, and transformed mouse embryo fibroblast cell lines. We have introduced specific antisense oligonucleotides into human mammary tumor cell lines in vitro to analyze the role of individual PKC isoforms in radiation-induced cell death in breast cancer. MDA-MB-231 and MCF-7 cells treated with oligonucleotide directed against the PKC δ isoform exhibited impaired survival in response to 5.6 Gy γ-radiation as measured by mitochondrial metabolism of tetrazolium dye. The role of PKC δ in the breast tumor cell lines was of particular interest, because contradictory reports exist in the literature regarding the role of PKC δ in cell survival and apoptosis. A comparison of the effects of the PKC δ antisense oligonucleotide and a nucleotide scrambled version of this nucleotide revealed only the antisense oligonucleotide decreased cell survival. The PKC δ antisense oligonucleotide decreased cell survival after exposure to low (1.5 Gy) radiation doses and in the absence of radiation insult. We found 3 μm rottlerin, a selective PKC δ inhibitor, to reduce MCF-7 and MDA-MB-231 cell survival. Furthermore, MCF-7 cells transformed to express a dominant-negative mutant of PKC δ exhibited reduced survival. Comet analysis showed that PKC δ oligonucleotide treatment caused an accumulation of cells containing damaged DNA similar to that seen in 1.5 Gy radiation-treated cells. We conclude that PKC δ acts as a prosurvival factor in human breast tumor cells in vitro.