Objective: In various studies on isolated cardiomyocytes CO₂ is used to anesthetise the animals. The aim of the present study was to analyze the impact of anaesthesia with CO₂ on endothelin-1 (ET-1)- and noradrenaline (NA)- induced effects on isolated cardiomyocytes in contrast to a barbiturate-anaesthesia. Methods: We quantified (i) partial pressure of oxygen (pO₂) and CO₂ (pCO₂) and (ii) the pH-value by blood-gas analysis of arterial mouse blood. Furthermore, we analysed the effect of CO₂- vs. barbiturate-anaesthesia on the influence of hormones such as ET-1 and NA on (iii) amplitude and (iv) kinetics of calcium (Ca²⁺)-transients by Ca²⁺-fluorimetric analysis of isolated cardiomyocytes. Results: It was shown that anaesthesia of mice with CO₂ does not cause any significant hypoxia compared to the barbiturate-anaesthesia but causes a significant hypercapnia (pCO₂: 190 ±13 mmHg, n=6 vs. 41 ±9 mmHg, n=6) and a significant acidosis (pH: 6.8 ±0.06 vs. 7.1 ±0.02). However, these changes in pCO₂ and pH do not cause any significant effect either on the amplitude nor on the kinetics of Ca²⁺-transients in isolated ventricular (CO₂: n=22; barbiturate: n=22) or atrial (CO₂: n=20; barbiturate: n=20) cardiomyocytes. Also, the effects of the hormones ET-1 and NA are not influenced by the anaesthesia with CO₂. Conclusion: These results demonstrate, that CO₂-anesthesia of mice does not alter the hormone modulated Ca²⁺-transients in isolated cardiomyocytes. However, we noticed, that CO₂-anesthesia of mice caused significant hypercapnia and acidosis. Therefore, the application of CO₂ as a narcotic gas for laboratory animals should be assessed as being critical since other factors than tested here could be altered by the changes in the blood gases.