A sensitive fluorometric assay was developed for the enumeration of cells in microtiter plates. This assay is based on the fluorescence enhancement of propidium iodide (PI) upon binding with double-stranded nucleic acids. This fluorochrome is compatible with a wide range of reagents commonly used in the laboratory, thus washing the cells before staining is not necessary. PI, together with Triton-X 100 and EDTA, was added directly to the cell culture. After 16-18 h incubation at room temperature, intensity of fluorescence was determined with a micro-plate fluorometer. This quick and simple method is sensitive for as little as 1.95 x 10(3) mononuclear leukocytes, and provides a linear correlation (r = 0.999) between cell number and fluorescence up to 1 x 10(6) cells. Since PI has a large Stokes shift with excitation wavelength at common visible range and emission wavelength far out in the red region of the spectrum, it allows simultaneous detection of DNA and other fluorescent compounds such as calcein and fluorescein. This assay may prove to be a valuable alternative for cell number determination.