A caspase-mediated release of the 40-kDa catalytic fragment of the δ isoform (CF-δ) of protein kinase C (PKC-δ) is involved in apoptosis, but its actual role in apoptosis development is still unknown. In an effort to understand this role, we have used polyomavirus-transformed pyF111 rat fibroblasts, which are hypersusceptible to apoptosis as they constitutively hyperexpress PKC-δ, but cannot make the antiapoptotic Bcl-2 and Bcl-X_L proteins, while making the proapoptotic Bax protein. Calphostin C is reportedly both a specific inhibitor of PKC-δ activity (C. Keenan, N. Goode, and C. Pears, 1997, FEBS Lett. 415, 101–108) and an effective apoptogen (M. Murata et al., 1997, Cell. Mol. Life Sci. 53, 737–743). Exposure of pyF111 cells to calphostin C (75 nM) stimulated the translocation of the PKC-δ holoenzyme (holo-PKC-δ) onto the cytoplasmic particulate (CP) fraction between 15 and 45 min, which was after the release of mitochondrial cytochrome c but before the activation of cytoplasmic DEVD-specific caspases. The CF-δ fragment started accumulating only between 2 and 4 h, while apoptosis occurred mostly within 6 h. Incubating pyF111 cells with the much slower acting, apoptogenic topoisomerase-II inhibitors etoposide (VP-16) and teniposide (VM-26) also caused within 6 h a doubling of the CP-bound holo-PKC-δ-related activity but with no significant translocation of the holoenzyme to the CP fraction. Again this occurred after the release of cytochrome c but before the activation of DEVDases and the accumulation of the CF-δ. However, while calphostin C did not affect the δ-related activity in the nuclear membrane (NM) and nucleoplasmic (NP) fractions, VP-16 and VM-26 caused a prompt, large, and irreversible drop in the δ activity at the NM and a transient surge followed by a fall in the NP-associated activity. Hence, a surge of CP-anchored holo-PKC-δ activity is a common part of the signals given by various apoptogenic drugs to pyF111 cells. On the other hand, inhibition of δ-related activity, first at the NM and then in the NP fraction, is a specific feature only of the signals given by apoptogenic DNA-damaging agents.