The aim of this study was to investigate the staining characteristics of Victoria Blue B in alcohol solutions. Cytological specimens (liver and spleen tissue imprints, blood smears) were stained with methanol solutions of commercially available Victoria Blue B-Cl and with pure Victoria Blue B-BF4. The dye concentration, staining time, and protone concentration of the dye solution were varied. The dye solutions were characterized using spectrophotometry and thin-layer chromatography. Cytophotometry and image analysis were used to quantitate the staining pattern of cell nuclei. Feulgen-stained slides were used as controls. Victoria Blue B-BF4 gave excellent nuclear staining exhibiting a quantitative dye-substrate relationship, whereas commercial dyes resulted in lower staining intensity and less distinct nuclear texture. Dye concentration and staining time were, over wide ranges, not of critical importance for the quality of the staining. Under certain staining conditions, only cell nuclei were stained, with the background remaining completely unstained. We presume that, in alcohol solutions, Victoria Blue dye binds as a neutral dye molecule in conjunction with its anion. Victoria Blue B-BF4 staining provides a simple and reproducible staining technique for cytology which is suitable for use in automated cell-pattern recognition.