Perlecan is primarily a heparan sulfate containing proteoglycan found in all basement membranes. Rotary shadowed images of perlecan show it to contain three glycosaminoglycan (GAG) side chains extending from one end of its core protein. Domain I is at the N terminus of perlecan and contains three closely spaced Ser-Gly-Asp sequences that may serve in GAG attachment. We evaluated the serines in these three sequences for GAG attachment by preparing a cDNA construct encoding for the N-terminal half (domains I, II, and III) of perlecan and then a series of constructs containing deletions and mutations within domain I of the domain I/II/III construct, expressing these constructs in COS-7 cells, and then analyzing the recombinant product for GAG side chains and GAG type. The results showed that all three serine residues in the Ser-Gly-Asp sequences in domain I can accept both chondroitin and heparan sulfate side chains but that a cluster of acidic residues N-terminal to these sequences is the primary determinant responsible for targeting these sites for heparan sulfate. Furthermore, there are two elements that can enhance heparan sulfate synthesis at a targeted site: 1) the presence of a the SEA module in the C-terminal region of domain I and 2) the presence of multiple acceptors in close proximity. These results indicate that the proportion of heparan and chondroitin sulfate at any one site in domain I of perlecan is regulated by multiple factors.