Intracellular free calcium concentrations (Ca++i) were studied in polymorphonuclear leukocytes (PMNs) from 13 athyreotic patients who had been previously treated by total thyroidectomy and radioiodine therapy for differentiated thyroid carcinoma, and from age- and sex-matched euthyroid healthy controls. Patients were studied twice, when hypothyroid (visit 1) and after restoration of euthyroidism by L-T4 TSH-suppressive therapy (visit 2). PMNs from patients at visit 1 had significantly lower resting (Ca++)i levels compared to both visit 2 and controls. Values at visit 2 did not differ from those of the controls. Stimulus-induced (Ca++)i rise was also significantly blunted at visit 1 and normalized at visit 2, possibly through a differential contribution of distinct intracellular Ca++ stores, as suggested by the response pattern to the chemotactic agent, N-formyl-Met-Leu-Phe (fMLP), to the selective SERCA pump inhibitor, thapsigargine, and to the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP). In vitro treatment of PMNs from healthy subjects with high TSH concentrations impaired intracellular Ca++ store function. Both resting (Ca++)i levels and fMLP-induced (Ca++)i rise increased in the presence either of low-concentration TSH or of T4, but effects of TSH and T4 were not additive. T3, rT3, and TRIAC had no effect. In conclusion, this study provides evidence for a direct relationship between thyroid status and (Ca++)i homeostasis in human PMNs, mainly related to direct actions of TSH and T4 on these cells.