Cholesterol from peripheral tissue, carried by HDL, is metabolized in the liver after uptake by the HDL receptor, SR-B1. Hepatocytes have long been considered the only liver cells expressing SR-B1; however, in this study we describe two disparate immunofluorescence (IF) experiments that suggest otherwise. Using high-resolution confocal microscopy employing ultrathin (120 nm) sections of mouse liver, improving z-axis resolution, we identified the liver sinusoidal endothelial cells (LSEC), marked by FcγRIIb, as the cell within the liver expressing abundant SR-B1. In contrast, the hepatocyte, identified with β-catenin, expressed considerably weaker levels, although optical resolution of SR-B1 was inadequate. Thus, we moved to a different IF strategy, first separating dissociated liver cells by gradient centrifugation into two portions, hepatocytes (parenchymal cells) and LSEC (non-parenchymal cells). Characterizing both portions for the cellular expression of SR-B1 by flow cytometry, we found that LSEC expressed considerable amounts of SR-B1 while in hepatocytes SR-B1 expression was barely perceptible. Assessing mRNA of SR-B1 by real time PCR we found messenger expression in LSEC to be about 5 times higher than in hepatocytes.