T cell surface sialylation changes during maturation in the thymus. We have previously demonstrated increased expression of mRNA encoding the Gal beta 1, 3GalNAc alpha 2,3-sialyltransferase in mature medullary human thymocytes, compared with immature cortical thymocytes. For this enzyme, increased expression of transferase mRNA correlated with increased sialylation of O-glycans. We have now examined the pattern of expression in the human thymus of two additional sialyltransferases, the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase (ST6N) and the Gal beta 1,3/4GlcNAc alpha 2,3-sialyltransferase (ST3N). The patterns of mRNA expression were compared with the pattern of binding of two sialic acid-specific plant lectins, Sambucus nigra agglutinin and Maackia amurensis agglutinin, which preferentially recognize alpha 2,6- and alpha 2,3-linked sialic acids, respectively, on N-glycans. By in situ hybridization, mRNA encoding ST3N was detected uniformly throughout the thymus. All thymocytes bound M. amurensis agglutinin, demonstrating a direct correlation between the level of ST3N mRNA expression and cell-surface glycosylation. In contrast, mRNA encoding ST6N was also expressed uniformly throughout the thymus; however, only mature (CD3hi) medullary thymocytes bound S. nigra agglutinin. On mature thymocytes, S. nigra agglutinin appeared to bind primarily to the cell-surface glycoprotein CD45; since only the mature thymocytes expressed the CD45RA isoform, while both mature and immature populations expressed the CD45R0 isoform, CD45RA may be a preferred substrate for ST6N. These results demonstrate that glycoprotein sialylation is tightly regulated during T cell development and that the developmentally regulated expression of specific oligosaccharide structures on the cell surface may be influenced by expression of both the relevant glycosyltransferase and specific acceptor substrates.