The present study was designed to assess the influence of antigen retrieval and/or DNA denaturation on the quantitative estimation of bromodeoxyuridine (BrdU) in formalin-fixed paraffin-embedded tissue. Specimens of small intestine from rats injected with BrdU were routinely fixed and embedded in paraffin. For antigen retrieval, sections were pretreated with microwave irradiation or enzymatically (pepsin or trypsin). Acid hydrolysis was used as a DNA denaturation method. Immunostaining of BrdU-labeled cells was performed. The best results, regarding tissue morphology and immunostaining, were obtained with microwave pretreatment followed by acid hydrolysis. Enzymatic pretreatment resulted in damage of tissue morphology and/or high background staining. Microwave alone, without DNA denaturation, resulted in a lower percentage of BrdU positive cells. The significance of validation studies is emphasized when the level of positivity for a prognostic marker, such as BrdU, is assessed.