BACKGROUND Maintenance of extracellular calcium (Ca) concentration within narrow limits is critical for normal cell function and optimization of bone health. Ionized Ca (iCa), the form of Ca that is regulated, has been reported to vary inversely with blood alcohol concentration (BAC). The mechanism by which ethanol decreases blood iCa levels is unknown. However, one possible explanation is that it is, at least partially, a methodological artifact due to an effect of ethanol on the function of the ion selective electrode used to measure iCa. The purpose of this study was to determine if ethanol interferes with the measurement of iCa and if this interference can account for the typically observed in vivo effects of ethanol consumption on blood iCa levels. METHODS Ethanol (0-5 microl/ml) was added to blood or iCa standards and the iCa concentration measured using the ICA2 iCa analyzer (Radiometer) or the I-Stat Clinical Analyzer (Abbott Laboratories). Both instruments use an ion selective electrode to measure iCa. The relationship between iCa and BAC determined from the ex vivo addition of ethanol to blood was compared with relationships obtained in vivo during chronic ethanol consumption. RESULTS Addition of ethanol to blood or iCa standards resulted in a dose-dependent decrease in iCa concentration when iCa was measured using the ICA2 analyzer but had no effect on iCa concentration when measured with the I-Stat Clinical Analyzer. Ethanol's effect on iCa with the ICA2 analyzer did not differ between blood and standards, and ethanol had no effect on pH, suggesting that the ethanol-induced decrease in iCa was due to a methodological artifact. However, the magnitude of ethanol's effect was small and does not account entirely for the relationship between iCa and BAC observed in vivo with chronic ethanol consumption. CONCLUSIONS Ethanol can interfere with the measurement of iCa using ion selective electrodes, but this interference depends on the analyzer used. This is a significant methodological issue that has largely been unrecognized in the field of alcohol research. Although this interference does not explain entirely the relationship between iCa and BAC observed in vivo in studies on chronic ethanol consumption, it complicates investigations designed to assess the effect of ethanol on Ca homeostasis.