—To investigate the in vivo role that hepatic lipase (HL) plays in HDL metabolism independently of its lipolytic function, recombinant adenovirus (rAdV) expressing native HL, catalytically inactive HL (HL-145G), and luciferase control was injected in HL-deficient mice. At day 4 after infusion of 2×10⁸ plaque-forming units of rHL-AdV and rHL-145G-AdV, similar plasma concentrations were detected in postheparin plasma (HL=8.4±0.8 μg/mL and HL-145G=8.3±0.8 μg/mL). Mice expressing HL had significant reductions of cholesterol (−76%), phospholipids (PL; −68%), HDL cholesterol (−79%), apolipoprotein (apo) A-I (−45%), and apoA-II (−59%; P<0.05 for all), whereas mice expressing HL-145G decreased their cholesterol (−49%), PL (−40%), HDL cholesterol (−42%), and apoA-II (−89%; P<0.005 for all) but had no changes in apoA-I. The plasma kinetics of ¹²⁵I-labeled apoA-I HDL, ¹³¹I-labeled apoA-II HDL, and [³H]cholesteryl ester (CE) HDL revealed that compared with mice expressing luciferase control (fractional catabolic rate [FCR] in d⁻¹: apoA-I HDL=1.3±0.1; apoA-II HDL=2.1±0; CE HDL=4.1±0.7), both HL and HL-145G enhanced the plasma clearance of CEs and apoA-II present in HDL (apoA-II HDL=5.6±0.5 and 4.4±0.2; CE HDL=9.3±0.0 and 8.3±1.1, respectively), whereas the clearance of apoA-I HDL was enhanced in mice expressing HL (FCR=4.6±0.3) but not HL-145G (FCR=1.4±0.4). These combined findings demonstrate that both lipolytic and nonlipolytic functions of HL are important for HDL metabolism in vivo. Our study provides, for the first time, in vivo evidence for a role of HL in HDL metabolism independent of lipolysis and provides new insights into the role of HL in facilitating distinct metabolic pathways involved in the catabolism of apoA-I– versus apoA-II–containing HDL.