The pulmonary microvasculature has a high capacity for transendothelial albumin transport. Recent in vitro data from our laboratory suggest that inflammatory mediators such as thrombin may amplify transendothelial albumin transport by acid sphingomyelinase-dependent recruitment of caveolin-1 to lipid rafts. Here, we tested this novel concept in the intact microvasculature of the isolated blood-perfused rat lung. Endothelial cells were labeled in situ with fura-2 while microvessels were perfused with FITC-labeled albumin (1 mg/ml). Endothelial uptake of albumin was quantified as ratio of fluorescence intensities determined at 470 nm and 360 nm after 60 min. Compared to control, thrombin (50 U/ml) increased the pulmonary microvascular FITC/fura-2 ratio from 0.43±0.06 to 1.37±0.29 (n=5 each, p<0.05). This effect was attributable to enhanced FITC intensity (p<0.05) as fura-2 intensity remained unchanged. Line profiles of fluorescence intensities perpendicular to the vascular axis localized FITC fluorescence to endothelial cells and the subendothelial space. Inhibition of acid sphingomyelinase by imipramine (10 &mumol/l) prevented the thrombin-induced increase in the microvascular FITC/fura-2 rati (0.41±0.04, p<0.05 vs. thrombin w/o imipramine). Hence, thrombin stimulates transcellular albumin transport in a sphingomyelinase-dependent fashion in intact lung microvessels.