Aims: This study addressed the hypotheses that the hypercapnic flow response in wild-type (WT) mouse heart is mainly mediated by nitric oxide (NO) and, thus, severely blunted in endothelial nitric oxide synthase knockout (eNOS-KO) mice and in WT mice after continuous pharmacological block (2 weeks) of NOS enzymes (WT-LN). Methods and results: Step changes of arterial pCO ₂ were performed in isolated perfused hearts (n = 105). Contributions of NOS (L-NAME, TRIM), cyclooxygenase (indomethacin), epoxyeicosanotrienes (miconazole), adenosine A_2A-receptors (SCH 58261), K _V-channels (4-AP), K_Ca-channels (TEA), and K _ATP-channels (glibenclamide) were studied in WT and eNOS-KO mouse hearts. Change of arterial pCO₂ increased coronary flow by 31.3 ± 4% in WT, a response that was significantly decreased to 9.2 ± 6% after L-NAME. Additional glibenclamide infusion (n = 5) completely abolished the steady-state flow increase during hypercapnic acidosis (-4.2 ± 2.3%, P = 0.004 vs. control). Hearts from eNOS-KO mice as well as WT-LN showed a fully preserved flow response insensitive towards NOS-blockade. Whereas indomethacin, miconazole, TEA, and SCH 58261 were ineffective to reduce the flow response, glibenclamide blunted it in eNOS-KO hearts. Conclusion: NO-production and K _ATP-channel activation together may fully account for the steady-state hypercapnic flow response in mouse heart. However, chronic deletion of eNOS does not result in a reduced hypercapnic flow response. Enhanced activation of K_ATP-channels and potentially K_v-channels contributes to the compensatory mechanisms involved in the hypercapnic flow response when eNOS activity is absent.