To identify immunoglobulin (Ig)-producing cells with immunohistochemistry, conventional methods of preparation using chemical fixatives have problems such as the artificial diffusion of components and antigen masking. The "diffusion artifact" is caused by the translocation of soluble proteins like Ig from the serum to cytoplasm or vice versa. We have examined the immunolocalization of serum proteins, such as Ig kappa light chain (Igkappa), IgG1 heavy chain (IgG1), and albumin, in immunized mouse spleens after a peritoneal injection of human hemoglobin. Better preservation of morphology and immunoreactivity was obtained with the "in vivo cryotechnique" (IVCT) followed by freeze-substitution, than with conventional preparative methods. Although Ig-producing cells were not clearly detected in red pulp of 2-day-immunized spleens with the conventional methods, Igkappa-immunopositive cells with rich cytoplasm were detected in the red pulp with IVCT, especially in the subcapsular and peritrabecular areas, where IgG1-immunopositive cells were rarely observed. In 7-day-immunized spleens prepared with IVCT, Igkappa- or IgG1-immunopositive cells were mostly located in peritrabeculae. The development of Ig-producing cells was clarified in the specimens prepared with IVCT, which proved to be useful for analyzing the native morphology and distribution of Ig-producing cells.