We investigated the function of the Ca 2/ -dependent protein kinase C (PKC) b 1 in the regulation of endothelial barrier property. Human dermal microvascular endothelial cells (HMEC-1) were transduced with full-length PKCb 1 antisense (AS) cDNA or control pLNCX vector to generate stable cell lines (HMEC-AS and HMEC-pLNCX, respectively). Analyses indicated that HMEC-AS expressed the antisense PKCb 1 transcript with decreased PKCb protein level (without a change in PKCa or PKCe). The baseline transendothelial 125 I-albumin clearance rates of HMEC-1, HMEC-pLNCX, and HMEC-AS were 5.0 { 0.5 1 10 02 , 6.8 { 0.4 1 10 02 , and 6.9 { 0.6 1 10 02 ml/min, respectively. Activation of HMEC-1 and HMEC-pLNCX with phorbol 12-myristate 13-acetate (PMA) increased the rates to the respective 14.5 { 1.7 1 10 02 ml/min and 16.9 { 2.8 1 10 02 ml/min (corresponding to 191% and 149% increases over baseline). However, in HMEC-AS, PMA increased the rate to 9.8 { 1.0 1 10 02 ml/min (42%). When HMEC-1 and HMEC-pLNCX were activated with thrombin, the rates increased to 10.8 { 1.4 1 10 02 and 14.0 { 1.9 1 10 02 ml/min, respectively (116% and 106%). In contrast, thrombin stimulation of HMEC-AS more than doubled the increase to 27.2 { 3.5 1 10 02 ml/min (294%). Furthermore, the thrombin-induced peak increase in the [Ca 2/ ] i in HMEC-AS was greater than in control cells. Fluorescence-activated cell sorter analysis of thrombin receptor expression indicated that the augmented thrombin-induced responses were not attributable to altered receptor density in HMEC-AS. These results indicate that PKCb functions in a negative feedback manner to inactivate thrombin-generated signals and thereby modulates the endothelial permeability increase. Because decreased PKCb expression significantly reduced the PMA-induced permeability increase, PKCb may downregulate thrombin receptor function upstream of PKC activation (i.e., Ca 2/ ).