Abstract Cytoplasmic free calcium ([Ca 2+] i) is a key intracellular messenger in many cell types. We have used fluo-3, a recently developed calcium probe, to study [Ca 2+] i in resting and stimulated human peripheral blood T lymphocytes. The spectral properties of fluo-3 permit analysis of [Ca 2+] i in flow cytometers with a 488 nm argon laser excitation source and fluorescein filter settings. The data obtained with fluo-3 are both qualitatively and quantitatively in good agreement with the data in the literature. After stimulation of T lymphocytes with the mitogens phytohaemagglutinin, concanavalin A and with OKT3, and anti-CD3 monoclonal antibody, a biphasic [Ca 2+] i response was observed, with an early EGTA-insensitive [Ca 2+] i rise, followed by an EGTA-sensitive sustained [Ca 2+] i plateau. Non-mitogenic monoclonal antibodies directed against the CD5, CD28 and CD7 T cell surface antigens elicited [Ca 2+] i rises only when crosslinked on the cell surface with goat anti-mouse IgG. Conversion of fluorescence data into absolute [Ca 2+] i values by means of a non-disruptive calibration procedure, yielded a [Ca 2+] i of 107 ± 18 nM (mean ± SD, n = 13) in resting T lymphocytes. Time-dependent loss of cellular dye content limits the precision of the calibration procedure in experiments of longer duration. We conclude that fluo-3 promisingly extends the potential application field of flow cytometers with 488 nm argon lasers to [Ca 2+] i studies in T lymphocytes.