Perlecan is a multidomain proteoglycan, usually substituted with heparan sulphate (HS), and sometimes substituted with both HS and chondroitin sulphate (CS). In this paper, we describe perlecan purified from HEK-293 cells substituted with HS, CS and keratan sulphate (KS). KS substitution was confirmed by immunoreactivity with antibody 5D4, sensitivity to keratanase treatment, and fluorophore-assisted carbohydrate electrophoresis. HEK-293 perlecan failed to promote FGF-dependent cell growth in an in vitro assay. This study is the first to report perlecan containing KS, and makes perlecan one of only a very few proteoglycans substituted with three distinct types of glycosaminoglycan chains.