Background: Although alcohol septal ablation (ASA) is increasingly used in hypertrophic cardiomyopathy (HC) patients who are refractory to medical therapy, the amount of alcohol that is required has not been well studied. This study sought to determine the amount of alcohol that is necessary to achieve clinical benefits of ASA. Methods: Myocardial perfusion imaging was used to determine the size of the myocardial infarction produced by ASA in 54 HC patients. Left ventricular outflow gradients (LVOTg) were determined invasively before and after ASA and by Doppler echocardiography before and at a median of 3 months after ASA. Results: LVOTg decreased at rest and after provocation in response to ASA and this was maintained on follow-up at 3 months. There was no relationship between the amount of alcohol infused and the infarct mass as determined by myocardial perfusion imaging. While the infarct mass was not correlated with the drop in the LVOTg at rest or with provocation, the quantity of alcohol infused was correlated with the drop in LVOTg at rest (r = 0.27, p = 0.05) and with provocation (r = 0.34, p = 0.02). Furthermore, infusing more than 2ml of absolute alcohol was associated with a drop in the LVOTg by more than 60 mmHg at rest (p = 0.02) and by more than 130 mmHg with provocation (p = 0.05). Conclusions: Although lower amounts of alcohol infusion are desirable to avoid side-effects, it might be prudent to infuse around 2ml of absolute alcohol in order to achieve the desirable degree of LVOTg reduction in ASA.