The effect of penicillin on the human peripheral lymphocytes was studied by steady fluorescent technique and ratiometric fluorescence dye, Fura-2. The change of the free calcium concentration in cytosol was examined under different conditions. A characterization of Fura-2-Ca interaction in an isotonic saline solution showed that Ca2+ formed a 1:1 Fura-2-Ca complex with the apparent dissociation constant 1.81×10-7 mol/L. The mechanism, by which penicillin induced the decrease of [Ca2+]i, was discussed in detail. The low dose of penicillin might modify the lymphocytes' immunology response by interfering the increase in the intracellular free calcium concentration.