We measured the hydraulic conductivity (Lp) of the extracellular matrix (ECM) obtained after detaching bovine pulmonary microvascular endothelial (BPMVEC) and bovine pulmonary arterial endothelial cell (BPAEC) monolayers from the ECM at different days postseeding. From day 1 to day 5 in culture, the total Lp (i.e., of cell monolayer + ECM) decreased from basal values of 17.1 +/- 4.0 to 8.5 +/- 1.6 x 10(-6) cm.s-1.cmH2O-1 in BPAEC (P < 0.05) and 7.6 +/- 1.1 to 3.7 +/- 0.8 in BPMVEC (P < 0.05), respectively, and on day 5 the total Lp values were lower in BPMVEC than in BPAEC (P < 0.05). On the 5th day, ECM Lp was 55.0 +/- 8.3 in BPAEC and 10.7 +/- 0.9 cm.s-1.cmH2O-1 in BPMVEC (P < 0.05), indicating that the contribution of ECM to the total Lp was greater in BPMVEC than in BPAEC. Treatment of [3H]acetate-labeled ECM with Streptomyces hyaluronidase (HAse; 6 U/ml for 10 min) released sixfold greater radioactivity in BPMVEC compared with untreated BPMVEC controls; a similar treatment of BPAEC did not release detectable radioactivity indicative of a higher hyaluronan content in the BPMVEC ECM. HAse treatment reduced the differences in total Lp between BPMVEC and BPAEC at different days postseeding. Moreover, on the 5th day after seeding, the ECM Lp of BPMVEC increased to a greater extent after HAse treatment than the ECM of BPAEC. These data indicate that the hyaluronan component of the ECM is an important determinant of the endothelial liquid-exchange barrier.