Promotion of JB6 epidermal cells to anchorage-independent growth requires exposure to TPA for greater than 4 days. Over a similar time span, a practically complete loss of enzymic and immunoreactive proteinkinase C (PKC) equivalents was observed at greater than 10 nM TPA. Promotion did not appear to require (transient) activation of PKC since PKC inhibitors H7 and HA1004 did not prevent but enhanced colony formation in soft agar at concentrations greater than IC50-values. The efficacy of the inhibitors in vivo was shown by their ability to suppress PKC-induced transcription of c-fos gen. PKC inhibitors that interfered with cell proliferation at lower concentrations than those required for PKC inhibition (sphingosine, staurosporin, sangivamycin, trifluoperazine) did not stimulate anchorage-independent growth. As H7 as well as HA1004 were able to promote JB6 cells in the complete absence of TPA, and induced neither depletion nor processing of PKC we postulate that depletion/inactivation rather than activation of PKC correlates with the promotion of epidermal JB6 cells to anchorage-independent growth.