Graves' disease (GD) is an autoimmune disease that involves aberrant B and T lymphocyte responses. Detailed knowledge about lymphocyte subpopulation composition will therefore enhance our understanding of the pathogenesis of GD and might support the development of new immunomodulatory treatment approaches. The aim of this study was to gain detailed insight into the composition of the peripheral blood lymphocyte compartment in GD before and during anti-thyroid drug therapy. Major B and T lymphocyte subpopulations were investigated by flow cytometry in peripheral blood from newly diagnosed GD patients (n = 5), GD patients treated with anti-thyroid drugs (n = 4), patients with recurrent GD (n = 7) and healthy controls (HC; n = 10). In GD patients, numbers of activated T lymphocytes [human leucocyte antigen D-related (HLA-DR)⁺ and CD25⁺] were increased. The B lymphocyte compartment in GD was characterized by significantly higher numbers of transitional (CD38(high) CD27⁻, P < 0.03) and pre-naive mature (CD38(low) CD27⁻ IgD⁺ CD5⁺, P < 0.04) B lymphocytes, while memory populations were slightly decreased. The increased numbers of CD5⁺, transitional and pre-naive mature B lymphocytes correlated positively with fT4 plasma levels. GD is associated with increased numbers of activated T lymphocytes and transitional and pre-naive mature CD5⁺ B lymphocytes within the peripheral blood. The increase in CD5⁺ B lymphocytes was due mainly to an increase in transitional and pre-naive mature B lymphocytes. Increased fT4 plasma levels might be associated with this increase in transitional and pre-naive mature CD5⁺ B lymphocytes.