Glycosaminoglycans in the form of heparan sulfate proteoglycans (HSPG) and chondroitin sulfate proteoglycans (CSPG) are required for normal kidney organogenesis. The specific roles of HSPGs and CSPGs on ureteric bud (UB) branching morphogenesis are unclear, and past reports have obtained differing results. Here we employ in vitro systems, including isolated UB culture, to clarify the roles of HSPGs and CSPGs on this process. Microarray analysis revealed that many proteoglycan core proteins change during kidney development (syndecan-1,2,4, glypican-1,2,3, versican, decorin, biglycan). Moreover, syndecan-1, syndecan-4, glypican-3, and versican are differentially expressed during isolated UB culture, while decorin is dynamically regulated in cultured isolated metanephric mesenchyme (MM). Biochemical analysis indicated that while both heparan sulfate (HS) and chondroitin sulfate (CS) are present, CS accounts for approximately 75% of the glycosaminoglycans (GAG) in the embryonic kidney. Selective perturbation of HS in whole kidney rudiments and in the isolated UB resulted in a significant reduction in the number of UB branch tips, while CS perturbation has much less impressive effects on branching morphogenesis. Disruption of endogenous HS sulfation with chlorate resulted in diminished FGF2 binding and proliferation, which markedly altered kidney area but did not have a statistically significant effect on patterning of the ureteric tree. Furthermore, perturbation of GAGs did not have a detectable effect on FGFR2 expression or epithelial marker localization, suggesting the expression of these molecules is largely independent of HS function. Taken together, the data suggests that nonselective perturbation of HSPG function results in a general proliferation defect; selective perturbation of specific core proteins and/or GAG microstructure may result in branching pattern defects. Despite CS being the major GAG synthesized in the whole developing kidney, it appears to play a lesser role in UB branching; however, CS is likely to be integral to other developmental processes during nephrogenesis, possibly involving the MM. A model is presented of how, together with growth factors, heterogeneity of proteoglycan core proteins and glycosaminoglycan sulfation act as a switching mechanism to regulate different stages of the branching process. In this model, specific growth factor-HSPG combinations play key roles in the transitioning between stages and their maintenance.