Cell number is usually evaluated during in vitro studies to estimate metabolic or pharmacological effects of specific compounds. However, estimation of in vitro cell density by direct cell counting is a laborious and time-consuming task, whereas indirect methods for cell quantitation have serious disadvantages such as environmental costs or inaccuracies derived from non-specific interferences. We developed a new method for in vitro cell density quantitation which employs carmine, a natural dye widely used for chromosome staining in cytological studies. Normal or transformed murine fibroblasts, avian normal fibroblasts, human epithelial HeLa cells, and insect cells, inoculated at a range of cell densities, were fixed with 4% formaldehyde/PBS and stained with 0.4% alcoholic-HCl carmine. The stain retained in cell monolayers was extracted with 0.01 M NaOH and spectrophotometrically measured at 531 nm. Invariably, high correlation coefficients between cell number and absorbance were obtained for each cell type, within a range of 5 x 10(3) to 5 x 10(5) cells. Moreover, identical cell growth curves were obtained when cell number was estimated over several days of culture by both direct cell counting and carmine staining methods. Our results show that the carmine staining method represents an easy, precise and reliable alternative for in vitro cell quantitation, avoiding interferences caused by cell components modulable by culture treatments, and over a wide range of cell types and cell densities.