Previous studies have shown that ethanol decreases the level of ionized calcium (iCa) in the blood, and appears to prevent a compensatory increase in parathyroid hormone level. We have shown, however, that the presence of ethanol interferes with the measurement of blood iCa by the most commonly used iCa analyzer. It is impossible to interpret ethanol-induced alterations in Ca-regulating hormone levels without accurate measurement of blood iCa, thus the purpose of this study was to determine if ethanol decreases blood iCa levels independent of methodological artifacts. The time course of ethanol's effect and the relationship between iCa and blood ethanol concentration (BEC) were also examined. Rats (n=22) received ethanol (1.5 or 3 g/kg body weight) or saline by intraperitoneal injection. Blood samples were obtained by tail nick at 0, 2, 6, 24, 48 h and 8 days postinjection, and analyzed for iCa, pH, and BEC. Blood iCa and pH were measured using the I-Stat Clinical Analyzer, whose performance is not affected by the presence of ethanol. Ethanol administration resulted in a decrease in blood iCa levels. The magnitude and time course of the decrease varied with dose of ethanol, being greater and more prolonged with the higher dose, and blood iCa levels were not fully recovered at 48 h postinjection. No significant relationship was found between individual iCa and BEC values. This study confirms that ethanol decreases blood iCa levels, independent of methodological artifacts. Prolonged disruptions in Ca homeostasis resulting from ethanol consumption could have implications for long-term bone health.