1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione (U-73122) has been proven to be a useful tool in investigation of phospholipase C (PLC)-coupled signal transduction during cell activation. In the present studies, the inhibition by U-73122 of cytosolic free Ca2+ concentration ([Ca2+]i) of neutrophils was investigated. U-73122 suppressed the [Ca2+]i elevation of neutrophils suspended in Ca(2+)-containing medium challenged by N-formyl-Met-Leu-Phe (fMLP), cyclopiazonic acid (CPA) and ionomycin. The concentrations of U-73122 required for inhibition of CPA- and ionomycin-induced changes with IC50 values 4.06 +/- 0.27 microM and 4.04 +/- 0.44 microM, respectively, is almost 10-times that required for inhibition of the fMLP-induced response (IC50 value 0.62 +/- 0.04 microM). U-73122 also reduced the intracellular Ca2+ mobilization of neutrophils suspended in Ca(2+)-free medium stimulated by fMLP and CPA, but not by ionomycin, with IC50 values 0.52 +/- 0.02 microM and 6.82 +/- 0.74 microM, respectively. 1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrr olidinedione (U-73343), a close analog of U-73122 that does not inhibit PLC activity, suppressed the [Ca2+]i elevation of neutrophils challenged by fMLP in Ca(2+)-containing medium, but not in Ca(2+)-free medium, with IC50 value 22.30 +/- 1.61 microM. In Mn(2+)-quench studies, U-73122 suppressed the Mn2+ influx in CPA-activated neutrophils (IC50 value was 7.16 +/- 0.28 microM) as well as in resting neutrophils (IC50 value was 6.72 +/- 0.30 microM). U-73343 also suppressed the Mn2+ influx in resting neutrophils in a concentration-dependent manner. These results suggest that the inhibitory effect of U-73122 on [Ca2+]i of activated neutrophils is attributed partly to the suppression of Ca2+ release from the intracellular Ca2+ stores through PLC inhibition, and partly to the blockade, especially at higher concentrations, of Ca2+ entry from the extracellular space through PLC-independent processes.