A new dimension has been added to multiparameter flow cytometric analysis through the recent development of techniques for rapidly measuring the fluorescence lifetime of probes bound to single cells. The lifetime measurements are made by phase-sensitive detection techniques in a flow cytometer (FCM) that also analyzes fluorescence intensity and other optical properties of stained cells. These lifetime assays have potential for elucidating the microenvironment of the interaction of fluorochrome probes and subcellular target molecules. Alterations in the lifetime of DNA probes have been observed in cells in different phases of the cell cycle, in different cell types, in differentiating cells, and in apoptotic cells with damaged chromatin. Lifetime differences noted also for intercalating dyes bound to DNA and dsRNA, indicated modifications in the modes of binding and provide the potential for analyzing both corformational states and nucleic acid metabolism. Future developments in the technology will provide multiple lifetime assays and thereby allow for detection and quantitation of selected subcellular probe-complexes with different lifetime signatures. These novel assays will expand the applications for quantitative studies on the binding of various chemical agents to DNA and other molecular targets in cells, and further improve methods for rapid screening of chemotherapeutic agents or environmentally toxic compounds.