1 Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca 2+ concentration ([Ca 2+ ] i by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca 2+ ] i peak in a concentration-dependent manner. 2 Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 mM) for 30 min attenuated the BK-induced IPs formation and Ca 2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3 The concentrations of PMA that gave half-maximal (pEC 50 ) inhibition of BK-induced IPs accumulation and an increase in [Ca 2+ ] i were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4a-phorbol 12,13-didecanoate at 1 mM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 mM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory e ect of PMA is mediated through the activation of PKC. 4 In parallel with the e ect of PMA on the BK-induced IPs formation and Ca 2+ mobilization, the translocation and down-regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against di erent PKC isozymes revealed that TECs expressed PKC-a, bI, bII, g, d, e, y and z. With PMA treatment of the cells for various times, translocation of PKC-a, bI, bII, g, d, e and y from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 h treatment caused a partial down-regulation of these PKC isozymes. PKC-z was not signi®cantly translocated and down-regulated at any of the times tested. 5 Treatment of TECs with 1 mM PMA for either 30 min or 6 h did not signi®cantly change the K D and B max receptor for BK binding (control: K D =1.7+0.3 nM; B max =50.5+4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory e ect of PMA on BK-induced responses. 6 In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca 2+ ] i increase or inhibit independently both responses to BK. The translocation of pKC-a, bI, bII, d, e, g, and y induced by PMA caused an attenuation of BKinduced IPs accumulation and Ca 2+ mobilization in TECs.