The canine 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 fused to GFP was stably expressed with a typical Golgi localization in MDCK II cells (MDCK II-PAPST1). The capacity for PAPS uptake into Golgi vesicles was enhanced to almost three times that of Golgi vesicles isolated from untransfected cells. We have previously shown that chondroitin sulfate proteoglycans (CSPGs) are several times more intensely sulfated in the basolateral than the apical secretory pathway in MDCK II cells (Tveit H, Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells. J Biol Chem. 280:29596-29603). Here we demonstrate that increased availability of PAPS in the Golgi lumen enhances the sulfation of CSPG in the apical pathway several times, while sulfation of CSPGs in the basolateral pathway shows minor changes. Sulfation of heparan sulfate proteoglycans is essentially unchanged. Our data indicate that CSPG sulfation in the apical pathway of MDCK II cells occurs at suboptimal conditions, either because the sulfotransferases involved have high K(m) values, or there is a lower PAPS concentration in the lumen of the apical secretory route than in the basolateral counterpart.