In the present work we have investigated the actions of the oxidizing sulfhydryl reagent thimerosal on different mechanisms which regulate intracellular free Ca 2+ concentration ([Ca 2+] i) in GH 4C 1 pituitary cells. In intact Fura-2 loaded cells, low concentrations of thimerosal potentiated the spike phase of the TRH-induced (thyrotropin-releasing hormone) rise in [Ca 2+] i, whereas high thimerosal concentrations inhibited it. The effect of thimerosal on the plateau phase was always inhibitory. The effect of thimerosal on the IP 3-induced calcium release (IICR) was studied in permeabilized cells using the Ca 2+ indicator Fluo-3. A low concentration of thimerosal (10 μM) stimulated IICR: the Ca 2+ release induced by 300 nM inositol-1,4,5-trisphosphate (IP 3) was enhanced in cells treated with thimerosal for 1 or 6 min (67 ± 11 nM and 34 ± 5 nM, respectively) as compared to control cells (17 ± 2 nM). On the other hand, a high concentration of thimerosal (100 μ inhibited IICR: when IP 3 (10 μM) was added after a 5 min preincubation with thimerosal, the IP 3-induced rise in [Ca 2+] i (46 ± 14 nM) was 57% smaller as compared with that seen in control cells (106 ± 10 nM). The effect of thimerosal on the voltage-operated Ca 2+ channels (VOCCs) was studied by depolarizing intact Fura-2 loaded cells by addition of 20 mM K + to the cuvette. The depolarization-evoked increase in [Ca 2+] i was inhibited in a dose-dependent manner by thimerosal. Direct evidence for an inhibitory effect of thimerosal on VOCCs was obtained by using the whole-cell configuration of the patch-clamp technique: thimerosal (100 μM) potently inhibited the Ba 2+ currents through VOCCs. In addition, our results indicated that thimerosal inhibited the caffeine-induced increase in [Ca 2+] i, and activated a capacitative Ca 2+ entry pathway. The actions of thimerosal were apparently due to its oxidizing activity because the effects were mostly reversed by the thiol-reducing agent dithiothreitol (DTT). We conclude that, in GH 4C 1 pituitary cells, the mobilization of intracellular calcium and the different Ca 2+ entry pathways are sensitive to redox modulation.