Drug overdose is a major cause of premature death among heroin users and there has been a call for studies to identify risk factors for fatal overdoses to improve preventive interventions. The majority of heroin related deaths involves consumption of heroin in combination with other drugs, ethanol being one of the drugs most commonly found. The concomitant use of ethanol and heroin may enhance the risk for a heroin overdose. It has been suggested that ethanol might inhibit the metabolism of heroin, however, the underlying biological mechanisms are poorly understood. This is the first preclinical study examining the effects of ethanol on the heroin metabolism. The aim of this study was to investigate whether ethanol modulates the metabolism of heroin in mice, thereby increasing the concentration of active metabolites (6MAM and/or morphine). A behavior test in mice was applied, where total distance travelled was registered after administration of ethanol in combination with heroin. The stimulating effect of heroin, measured in the locomotor activity test, reflects the concentration of active metabolites in the brain. In vivo and in vitro kinetic studies in blood and brain from mice were used to examine the concentration vs. time curves of heroin and heroin metabolites with and without the presence of ethanol. LC-MS/MS was used to quantify heroin and heroin metabolites in biological matrices. Our findings indicated that ethanol had small modulating effect on the metabolism of heroin. An elevated concentration of heroin in blood and brain was detected in the presence of ethanol, but no changes in the concentration vs. time curves of 6MAM or morphine in brain were found. These results were reflected in locomotor activity. Ethanol did not increase the maximum response, Emax, or result in prolonged locomotor activity.