Novel PKC isoforms - and - have recently been implicated in signaling by hypertonic stress. We investigated the role of the putative PKC- inhibitor, rottlerin, upon tonicity-dependent gene regulation. In the renal medullary mIMCD3 cell line, rottlerin blocked tonicity-dependent transcription of a TonE-driven luciferase reporter gene, as well as tonicity-dependent transcription of the physiological tonicity effector gene, aldose reductase, but not urea-dependent transcription. Consistent with these data, rottlerin inhibited tonicity-dependent expression of TonEBP at the mRNA and protein levels. Another inhibitor of both novel and conventional PKC isoforms, GF109203X, suppressed TonEBP-dependent transcription but failed to influence tonicity-inducible TonEBP expression. Global PKC down-regulation with protracted phorbol ester treatment, however, failed to influence tonicity-dependent signaling arguing against a PKC--dependent mechanism of rottlerin action in this model. In addition, hypertonic stress failed to induce phosphorylation of PKC-. Furthermore, in a PC-12 cell model with a comparable degree of tonicity-dependent transcription, constitutive overexpression of dominant negative-acting PKC- or PKC- effectively decreased tonicity signaling to ERK activation, as expected, but failed to influence TonE-dependent transcription. TonE-dependent transcription, however, remained rottlerin-sensitive in this PC-12 cell model. In aggregate, these data indicate that rottlerin dramatically inhibits tonicity-dependent TonEBP expression and TonE-dependent transcription but, despite its reputed mode of action, does so through a PKC--independent pathway.