We report functional differences in constitutive and agonist-mediated endothelial barrier function between cultured primary human umbilical vein endothelial cells (pHUVEC) and Clonetic cultured HUVEC (cHUVEC) grown in soluble growth factors and heparin. pHUVEC exhibited much lower basal transendothelial resistance (TER) than cHUVEC grown in medium supplemented with growth factors like basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), human epithelial growth factor (EGF) and heparin. Based on a numerical model of TER the increased basal TER in cHUVEC was due to effects on cell-matrix adhesion and membrane capacitance. Basic FGF and heparin increased constitutive TER in cultured pHUVEC, and heparin mediated additional increases in constitutive TER in pHUVEC supplemented with bFGF. EGF attenuated bFGF-mediated increases in TER. Unlike cHUVEC, both heparin and bFGF augmented TER through effects on cell-cell adhesion and membrane capacitance in pHUVEC based on the numerical model. Thrombin mediated quantitative greater amplitude and more sustained decline in TER in cultured cHUVEC than in cultured pHUVEC. Thrombin-mediated barrier dysfunction was attenuated in pHUVEC conditioned in EGF both in the presence or absence of heparin. Thrombin-mediated barrier dysfunction was also attenuated when monolayers were exposed to low concentrations of heparin, and there were additional attenuation in the presence of bFGF. cAMP stimulation mediated differential attenuation of thrombin-mediated barrier dysfunction between pHUVEC and cHUVEC. VEGF displayed differential effects in TER in serum-free medium. Taken together, these data demonstrate marked differential regulation of constitutive and agonist-mediated endothelial barrier function in response to mitogens and heparin stimulation.