The use of bisindolylmaleimide derivatives of staurosporine as selective inhibitors of protein kinase C (PKC) is in doubt following the report by Alessi [FEBS Lett. 402 (1997) 121-123] that Ro31-8220 and GF109203X are potent in vitro inhibitors of p70 S6 kinase and mitogen-activated protein kinase-activated protein kinase-1beta, as well as of PKC. Here we show that the phorbol ester-stimulated release of choline- and ethanolamine-metabolites from C6 glioma cells due to phospholipid hydrolysis by phospholipase D (PLD) is not inhibited by rapamycin or PD98059, specific inhibitors respectively of p70 S6 kinase and MAPKK (MEK) and thus of MAPKAP kinase-1beta but is still completely blocked by Ro31-8220. We conclude therefore that p70S6k and MAPKAP kinase-1beta as well as MAPK are not involved in signalling pathways downstream of PKC that regulate phorbol ester-stimulated phospholipid turnover and that the inhibitory action of Ro31-8220 occurs by blocking PKC which regulates at least one pathway to PLD activation. The PI-3 kinase inhibitor, wortmannin, inhibits the phorbol ester-stimulated release of ethanolamine- but not choline-metabolites from C6 cells suggesting that different PLD isoforms regulate the turnover of PtdEth and PtdCho in C6 cells. Both PLD isoforms are activated via PKC but the PtdEth-PLD is also regulated via a wortmannin-sensitive pathway.