Heparan sulfate (HS) has been proposed to be antiatherogenic through inhibition of lipoprotein retention, inflammation, and smooth muscle cell proliferation. Perlecan is the predominant HS proteoglycan in the artery wall. Here, we investigated the role of perlecan HS chains using apoE null (ApoE0) mice that were cross-bred with mice expressing HS-deficient perlecan ( Hspg2 Δ3/Δ3 ). Morphometry of cross-sections from aortic roots and en face preparations of whole aortas revealed a significant decrease in lesion formation in ApoE0/ Hspg2 Δ3/Δ3 mice at both 15 and 33 weeks. In vitro, binding of labeled mouse triglyceride-rich lipoproteins and human LDL to total extracellular matrix, as well as to purified proteoglycans, prepared from ApoE0/ Hspg2 Δ3/Δ3 smooth muscle cells was reduced. In vivo, at 20 minutes influx of human 125 I-LDL or mouse triglyceride-rich lipoproteins into the aortic wall was increased in ApoE0/ Hspg2 Δ3/Δ3 mice compared to ApoE0 mice. However, at 72 hours accumulation of 125 I-LDL was similar in ApoE0/ Hspg2 Δ3/Δ3 and ApoE0 mice. Immunohistochemistry of lesions from ApoE0/ Hspg2 Δ3/Δ3 mice showed decreased staining for apoB and increased smooth muscle α-actin content, whereas accumulation of CD68-positive inflammatory cells was unchanged. We conclude that the perlecan HS chains are proatherogenic in mice, possibly through increased lipoprotein retention, altered vascular permeability, or other mechanisms. The ability of HS to inhibit smooth muscle cell growth may also influence development as well as instability of lesions.