Immunostaining of cytologic preparations has been beset by problems of inconsistency, high background staining, and the requirement of different fixatives for different antigens. This study sought to identify a universal fixative and a simple fixation protocol suitable for a wide range of tissue antigens commonly employed for cytologic diagnosis. In an analysis of 23 fixation protocols involving acetone, acetone/methanol, acetone/formalin, glutaraldehyde, ethanol, methanol, and formal saline, fixation in 0.1% formal saline overnight at 27 degrees C followed by 10 min fixation in 100% ethanol produced the most consistent and optimal preservation of immunoreactivity which could be further enhanced by pre-treatment with microwaves for epitope retrieval. Blocking of endogenous peroxidase was not necessary with this fixation protocol. Provided the smears were well air-dried (for at least 14 hr) prior to immersion in formal saline, there was no need to employ adhesive-coated glass slides. The smears could be kept at 27 degrees C (room temperature) for at least 7 days and at -70 degrees C for 5 wk without loss of immunoreactivity as air-dried smears or after fixation in formal saline. One hundred percent acetone and 100% ethanol produced good morphology and immunoreactivity but a high level of background staining, whereas acetone-based mixtures resulted in inconsistent immunostaining.