Heparan sulfate (HS) binds with various proteins including growth factors, morphogens, and extracellular matrix molecules to regulate their biological functions. These regulatory interactions are considered to be dependent on the structure of HS, which is determined by HS sulfotransferases. To gain insights into the functions of HS sulfotransferases in the development of the nervous system, we examined the expression of these enzymes (3-O-sulfotransferase-1 [3-OST-1], -2, -4; 6-OST-1, -2, -3; and N-deacetylase /N-sulfotransferase-1 [NDST-1], -2, -3) by in situ hybridization and real-time reverse transcription-polymerase chain reaction (RT-PCR). The expression of these genes was spatiotemporally regulated. In the E16 cerebrum, the expression of these genes showed two patterns: (1) selective expression at cortical plate (CP) and ventricular zone (VZ) and (2) wider expression by the cells in the marginal zone (MZ), CP, subplate (SP), and VZ. At P1, most genes showed similar expression patterns, but after P7, these genes were expressed differentially in a layer-specific manner. In the P1 cerebellum, the external granule cell layer (EGL) expressed most genes, the expressions of which were down-regulated at P7. In contrast, Purkinje cells began to express many of these genes after P7. These complex expression patterns suggest that the structure of HS is altered spatiotemporally for regulating various biological activities in the developing brain including the proliferation of neuronal progenitors, extension of axons, and formation of dendrites. We discuss possible functional roles of these sulfotransferases in the signaling of several HS-binding proteins such as fibroblast growth factors, slit, netrin, and sonic hedgehog.