A new breath alcohol (ethanol) analyzer has been developed, which allows free exhalation, standardizes measured exhaled alcohol concentration to fully saturated water vapor at a body temperature of 37 °C (43.95 mg/L) and includes a built-in self-calibration system. We evaluated the performance of this instrument by comparing standardized alcohol concentration in freely expired breath (BrAC) with arterial (ABAC) and venous (VBAC) blood alcohol concentrations in fifteen healthy volunteers who drank 0.6 g of alcohol per kg body weight. The precision (coefficient of variation, CV) of the analyzer based on in vivo duplicate measurements in all phases of the alcohol metabolism was 1.7%. The ABAC/BrAC ratio was 2251 ± 46 (mean ± S.D.) in the post-absorptive phase and the mean bias between ABAC and BrAC × 2251 was 0.0035 g/L with 95% limits of agreement of 0.033 and -0.026. The ABAC and BrAC × 2251 were highly correlated (r = 0.998, p < 0.001) and the regression relationship was ABAC = 0.00045 + 1.0069 × (BrAC × 2251) indicating excellent agreement and no fixed or proportional bias. In the absorption phase, ABAC exceeded BrAC × 2251 by at most 0.04 ± 0.03 g/L when tests were made at 10 min post-dosing (p < 0.05). The VBAC/BrAC ratio never stabilized and varied continuously between 1834 and 3259. There was a proportional bias between VBAC and BrAC × 2251 (ABAC) in the post-absorptive phase (p < 0.001). The pharmacokinetic analysis of the elimination rates of alcohol and times to zero BAC confirmed that BrAC × 2251 and ABAC agreed very well with each other, but not with VBAC (p < 0.001). We conclude that this new breath analyzer using free exhalation has a high precision for in vivo testing. The BrAC reflects very accurately ABAC in the post-absorption phase and substantially well in the absorption phase and thereby reflects the concentration of alcohol reaching the brain. Our findings highlight the magnitude of arterio-venous differences in alcohol concentration and support the use of breath alcohol analyzers as a stand-alone test for medical and legal purposes. © 2006 Elsevier Ireland Ltd. All rights reserved.