Spatial information embedded in the extracellular matrix establishes the dorsoventral polarity of the Drosophila embryo through the ventral activity of a serine protease cascade. Pipe is a Golgi-localized protein responsible for generating this spatial information during oogenesis through sulfation of unknown glycans. Although Pipe has sequence homology to glycosaminoglycan 2-O-sulfotransferases, its activity and authentic substrates have not been demonstrated and genetic evidence has argued against a role for glycosaminoglycans in dorsoventral polarity establishment. Here, direct examination of matrix glycosaminoglycans demonstrates that pipe-mutant matrix shows decreased tri-sulfated heparan sulfate compared to wild-type matrix, with correspondingly increased 2-O-sulfated heparan sulfate. Chondroitin sulfate was not detected in this matrix. These results suggest that Pipe promotes 6-O- and/or N-sulfation of heparan sulfate but is not required for heparan sulfate 2-O-sulfation. We discuss the possible significance of these unexpected findings and how they might be reconciled with the genetic data.