To evaluate the effect of different tissue preservation, fixation and staining procedures on the expression of proliferation-associated Ki-67 antigen in cytologic samples to establish an easy and uniform way to handle cytologic specimens with an automated staining technique.Multiple touch imprints were made from eight breast tumors. The specimens were treated according to different protocols, and Ki-67 nuclear expression was compared to that in the corresponding histologic sections.In the unstained specimens, air drying at room temperature for up to four months or ethanol spray fixation preserved the material and offered excellent results. Processing effectively removed previous stain without additional chemical destaining. Antigen retrieval was not achieved in the previously Giemsa stained imprints and was suboptimal in those stained according to Papanicolaou.Immunocytochemical detection of Ki-67 is recommended for previously unstained cytologic specimens.