This study presents a quantitative comparison of the free cytoplasmic calcium concentration ([Ca2+]cyt) and the free concentration in the lumen of the dense tubules of the human platelet. The former was measured by the fluorescence of the high affinity indicator quin2 and latter by the fluorescence of chlorotetracycline (CTC). The CTC technique monitors calcium-CTC complex accumulation in the lumen of dense tubules and mitochondria when washed platelets were incubated in 2 mM Ca2+. Resting cytoplasmic and dense tubular Ca2+concentrations were studied in platelets from patients suffering from venous and arterial thrombosis. Compared with normal controls (0.40 ± 0.10, n = 54), the values of the calcium-CTC ratios were 0.68 ± 0.19 (n = 16, p< 0.005) in venous thrombosis; 0.75 ± 0.18 (N = 14, p< 0.005) in cardiovascular accident; 0.84 ± 0.18 (n = 6, p< 0.005) in occlu-sive peripheral vascular diseases; and 0.42 ± 0.10 (n = 21, p>0.1) in patient controls. The dense tubular Ca2+levels for both patients and controls were perfectly correlated with the cytoplasmic levels using an equation that assumes that the dense tubular free calcium concentration ([Ca2+]dt) has a second power dependence on [Ca2+]cytvl. The abnormal Ca2+handling of platelets obtained from thrombotic patients could be completely reversed by preincubation with the calcium channel blocker verapamil. These observations suggest that the primary Ca2+handling defect is the leakage through activated channels in the plasma membrane. The defect and the elevated resting [Ca2+]cytand [Ca2+]dtare adequate to explain the observation of increased rates of collagen-activated aggregation in the above-mentioned group of patients. The results can be explained by platelets from thrombosis patients being exposed to activating factors in the circulation, resulting in Ca2+channel activation. Channel activation persists through the process of platelet isolation and washing and is manifested in higher measured values of [Ca2+]cytand [Ca2+]dtin the “resting state.” This would bring the platelet closer to its aggregation when aggregation-inducing agents are added. The CTC test is shown to be a useful and convenient means of detecting this abnormality.