To delineate the extent of O‐galactosyltransferase deficiency within the lymphoid lineage, monoclonal antibody specific for the Thomsen‐Friedenreich (TF) antigen (Galβ1 → 3GalNAcα1‐O‐Ser/Thr) and its precursor the Tn antigen (GalNAcα1‐O‐Ser/Thr) were applied to the flow cytometric analysis of peripheral blood lymphocytes from a patient with permanent mixed‐field polyagglutinability (PMFP). We show that only a minor population of 4% expressed the Tn antigen which is in contrast to 93% of the patient's erythrocytes carrying the defect. Tn⁺ lymphocytes mainly belonged to the CD3⁺ subset, but were also CD19⁺ or CD16⁺. Both Tn⁺ and TF⁺ T cell clones from patient R. R. were established and shown to belong to the CD4⁺ or CD8⁺ antigenic subset. Three glycosyltransferase activities were determined in lysates from these clones: all Tn⁺ clones were deficient in UDP‐Gal: GalNAcα1‐O‐Ser/Thr β1 → 3 galactosyl‐transferase (β3Gal‐T) activity; by contrast this activity was present in all lysates from TF‐expressing clones. UDP‐GalNAc:polypeptide α‐N‐acetyl‐galactosaminyltransferase (GalNAc‐T) and UDP‐Gal:GlcNAc‐Rβ1 → 4 galactosyl‐transferase (β4Gal‐T) exhibited similar activities in both Tn⁺ and TF⁺ T cell clones. As a consequence of defective O‐galactosylation in Tn⁺ T cells, cell surface sialic acid of Tn⁺ clones was reduced by > 50% when compared to TF⁺ clones as demonstrated by sialic acid‐specific labeling using fluoresceinated Limax flavus agglutinin(LA) and flow cytometry. The Tn phenotype of T cell clones was stable for more than 1 year of continuous expansion in vitro. These data demonstrate that in PMFP, T cells may also be affected by the O‐galactosyltransferase deficiency which is accompanied by a substantial loss of cell surface sialic acid. However, the frequency of Tn⁺ lymphocytes in peripheral blood from patient R.R. was strikingly low. These T cell clones should be useful to study the defect at a genetic level and the importance of O‐linked carbohydrates for proper T cell function.