Activation of protein kinase c (PKC) reduces transcription from the polymerase III (pol III)-transcribed adenovirus VA gene. Data presented here support a role for PKC in disrupting the formation of transcription-competent initiation complexes. The study used the plasmids VA and VA/EL (VA gene with a linker to distinguish its transcript from that of the VA gene) in in vitro assays to show that preincubation of either template for a minimum of 10 min before the activation of PKC did not result in PKC-induced repression of transcription. In contrast, under the same conditions, efficient transcription occurs from a preincubated template but not from a second template if it is added during or after the activation of PKC. Simultaneous preincubation of both VA and VA/EL resulted in efficient transcription from both templates. Rescue experiments confirm that PKC modifies a target within transcription factor B (TFIIIB) because phosphocellulose fractionation of whole-cell extracts that yield partially purified pol III transcription factor, TFIIIB, successfully rescues VA transcription from PKC-induced repression. Subsequent studies confirmed that the TATA box-binding protein (TBP), a constituent of TFIIIB, substituted for the crude preparation of TFIIIB. These data support a conclusion that activation of PKC triggers a cascade that likely involves the sequestration or degradation of TBP, resulting in the disruption of the steps that leads to successful pol III transcription initiation.