A number of supravital fluorochromes are available to study lymphocyte homing in vivo. These include fluorescein isothiocyanate (FITC), which binds to cell surface proteins; Hoechst 33342, which binds to AT rich regions of cellular DNA; and the lipid bilayer incorporated dyes PKH-2 and PKH-26. The relative advantages and disadvantages of each of these probes for analyzing murine lymphocyte homing are as yet poorly understood. We evaluated the effects of each dye on labeling efficiency, as well as cell viability, homing, mitogen responsiveness and cytotoxicity. PKH-26 provided long-term labeling (up to 15 days) with the least detrimental effects on cellular function. Detection of FITC in vivo was impaired by tissue autofluorescence, and the dye acted as a co-mitogen for lectin or IL-2-induced proliferation. Hoechst 33342 eluted from cells over a few hours and inhibited lymphocyte proliferation. PKH-2 had detrimental effects on cell viability and resulted in the down-modulation of peripheral lymph node homing receptor expression. None of the probes interfered with the induction of cytotoxic lymphocytes by IL-2. While these fluorochromes are easy to use and provide powerful tools to analyze the lymphocyte localization in vivo, our experiments demonstrate that important limitations are imposed by each probe that need to be considered by investigators during the design and interpretation of experiments.