Proliferating cell nuclear antigen (PCNA) in a useful marker for monitoring cell proliferation in most species. The immunostaining of PCNA on tissue cryosections, however, has been hampered by a loss of PCNA immunoreactivity during the staining process. The need for both identifying the actual phenotypes of proliferating cells and differentiating them from other tissue components prompted us to establish reliable techniques for PCNA immunostaining on cryosections and to apply these to double immunostainings with other markers. We tested various fixing conditions for rat tissue cryosections and the effect derived from unmasking with pepsin digestion to restore PCNA immunoreactivity after fixation. For single immunostainings, the unmasking was effective in most fixing conditions tested. Particularly, 4% paraformaldehyde/0.05% glutaraldehyde fixation followed by 0.001% pepsin digestion resulted in the strongest immunoreactivity for PCNA and the best morphology, and was the first choice for double immunostainings with relatively stable second antigens. Alternatively, periodate-lysine-paraformaldehyde fixation was also applicable to other second antigens which are labile to the former treatment. These techniques can serve in the collection of important information from frozen tissues regarding the relationship between PCNA and other markers of interest which are usually susceptible to routine formalin and/or paraffin embedding.