A phase-sensitive cytometer has been developed that combines flow cytometry and fluorescence lifetime spectroscopy measurement principles to provide unique features for making frequency-domain lifetime measurements on free fluorophore (solution) and on fluorophore-labeled cells/particles in real time. No other instrument can quantify lifetimes directly and resolve heterogeneous fluorescence based on differences in lifetimes (expressed as phase shifts), while maintaining the capability to make conventional flow cytometric measurements. The technology has been characterized with respect to measurement precision, linearity, sensitivity, and dynamic range. Fluorescence lifetime distributions have been measured on autofluorescence lung cells, thymocytes labeled with antibody conjugated to fluorophores for studying fluorescence quenching as a function of antibody dilution and F/P ratio, cells stained with DNA-binding fluorochromes, and on particles labeled with fluorophores and free fluorophore (solution). Phase-resolved, fluorescence signal- intensity histograms have been recorded on thymocytes labeled with a phycoerythrin/Texas Red tandem conjugate and propidium iodide to demonstrate the resolution of signals from highly overlapping emission spectra. This technology adds a new dimension to flow analyses of free and cell/particle-bound fluorophore. Lifetimes can be used as spectroscopic probes to study the interaction of markers with their targets, each other, and the surrounding microenvironment.