GPIHBP1-deficient mice (Gpihbp1(-/-)) exhibit severe chylomicronemia. GPIHBP1 is located within capillaries of muscle and adipose tissue, and expression of GPIHBP1 in Chinese hamster ovary cells confers upon those cells the ability to bind lipoprotein lipase (LPL). However, there has been absolutely no evidence that GPIHBP1 actually interacts with LPL in vivo. Heparin is known to release LPL from its in vivo binding sites, allowing it to enter the plasma. After an injection of heparin, we reasoned that LPL bound to GPIHBP1 in capillaries would be released very quickly, and we hypothesized that the kinetics of LPL entry into the plasma would differ in Gpihbp1(-/-) and control mice. Indeed, plasma LPL levels peaked very rapidly (within 1 min) after heparin in control mice. In contrast, plasma LPL levels in Gpihbp1(-/-) mice were much lower 1 min after heparin and increased slowly over 15 min. In keeping with that result, plasma triglycerides fell sharply within 10 min after heparin in wild-type mice, but were negligibly altered in the first 15 min after heparin in Gpihbp1(-/-) mice. Also, an injection of Intralipid released LPL into the plasma of wild-type mice but was ineffective in releasing LPL in Gpihbp1(-/-) mice. The observed differences in LPL release cannot be ascribed to different tissue stores of LPL, as LPL mass levels in tissues were similar in Gpihbp1(-/-) and control mice. The differences in LPL release after intravenous heparin and Intralipid strongly suggest that GPIHBP1 represents an important binding site for LPL in vivo.