The subclasses of IgG antibodies formed by grass pollen-allergic patients during immunotherapy were investigated by using a radioallergosorbent test (RAST) and a quantitative immunofluorescence method known as the defined antigen substrate spheres (DASS) system. By the use of rabbit antisera directed against the subclasses of IgG, the specificity of which was checked in the passive hemagglutination and immunofluorescence techniques, it was shown that a relatively high proportion of the grass pollen-specific antibodies belonged to the IgG4 subclass. Apart from the high binding activity of IgG4 which increased during treatment, a moderate binding activity of the other subclasses was also found. Binding of all subclasses increased slightly in the pollen season and could be specifically blocked by perincubation with soluble grass pollen extract. The results of the IgG4 binding, determined in vitro with the DASS system, and the blocking activity of the sera, determined in vivo by skin tests are suggestive for a relation between these activities. Also in the group of patients with a low IgE-RAST score, the skin reactivity decreased as the IgG4 binding activity increased.