Protein kinase C (PKC) is involved in mediating the tonic component of gastrointestinal smooth muscle contraction in response to stimulation by agonists for G-protein coupled receptors. We present pharmacological and immunohistochemical evidence indicating that a member of the novel PKC (nPKC) isoforms, PKC, is involved in maintaining muscarinic receptor-coupled tonic contractions of the guinea-pig ileum. The tonic component of carbachol-evoked contractions was enhanced by the activator of conventional and nPKCs, phorbol 12,13-dibutyrate (PDBu, 200 nM or 1 µM) and by ingenol 3,20-dibenzoate (IDB, 100 nM or 500 nM), an activator of nPKCs. Enhancement was unaffected by concentrations of bisindolylmaleimide 1 (BIM-1, 22 nM) that block conventional PKCs or by a PKC-specific inhibitor peptide, but was attenuated by higher doses of BIM-1 (2.2 µM). Relevant proteins were localized at a cellular and subcellular level using confocal analysis. Immunohistochemical staining of the ileum showed that PKC was exclusively expressed in smooth muscles distributed throughout the layers of the gut wall. PKC-immunoreactivity was prominent in enteric neurons but was largely absent from smooth muscle of the muscularis externa. Treatment with PDBu, IDB or carbachol resulted in a time and concentration dependent translocation of PKC from the cytoplasm to filamentous structures within smooth muscle cells. These were parallel to, but distinct from, actin filaments. The translocation of PKC in response to carbachol was significantly reduced by scopolamine or calphostin C. The present study indicates that the tonic carbachol-induced contraction of the guinea-pig ileum is mediated through a nPKC, probably PKC.