Immunohistochemical detection of p53 in paraffin-embedded biopsy specimens has important diagnostic, prognostic and therapeutic applications. In this study the influence of four different methods of fixation on immunohistochemical staining have been investigated in breast tumor specimens. The application of antigen retrieval methods was examined and the results of two different methods of antigen unmasking were compared. Optimal results were obtained using samples fixed in 70% ethanol and Carnoy solution. Tissues fixed in neutral buffered formalin or Bouin solution required preliminary procedures for antigen unmasking. The technique of microwave antigen retrieval gave crisp, clear immunohistochemical staining for p53 in tissues fixed in neutral buffered formalin or Bouin solution. In conclusion, the findings in this study show that: 1. The most suitable fixatives for immunohistochemical detection of p53 are 70% ethanol and Carnoy solution. 2. Antigen retrieval procedures by microwave irradiation are recommended for tissues fixed in 10% neutral buffered formalin or in Bouin solution; this pre-treatment improves the quality of the immunohistochemical reaction and decreases the possibility of false negative results. 3. The cytoplasmic accumulation of p53 should be accepted as a fact, not as a result of poor fixation and should be assessed in parallel with the nuclear reaction.