Staining of thymus tissue with the plant lectin peanut agglutinin (PNA) is a classic technique for defining the cortical (PNA+) and medullary (PNA-) regions of this tissue. These two regions are primarily composed of immature and mature thymocytes, respectively. Conversion of the PNA+ to the PNA- phenotype has been attributed to masking of the cell surface carbohydrate receptors of PNA by sialic acid during the intrathymic maturation of these cells. We present evidence that the regulated expression of a single glycosyltransferase, a Gal beta 1,3GalNAc alpha 2,3-sialyltransferase, can account for this glycosylation change. This enzyme sialylates the preferred ligand of PNA, Gal beta 1,3GalNAc, forming the sequence NeuAc alpha 2,3Gal beta 1,3GalNAc, thus masking PNA binding sites. Expression of the enzyme is inversely proportional to expression of the PNA receptor, as evidenced by analysis of T-lymphoblastoid cell lines and by in situ hybridization experiments in human thymic tissue.