Botulinum toxin (BT) injection is an alternative treatment of achalasia. The aim of the study was to examine outcomes of patients treated with BT in the Czech Republic. Since 1997, 49 patients with achalasia have been treated with BT. We prospectively evaluated the effect of BT injection on 41 patients during a median follow-up of 24 months (range 9-62). Esophageal manometry was performed before and at 3-5 months after the injection. In 16 patients, BT was injected from the antegrade angle only (subgroup A), in 15 patients, BT was injected from both retrograde and antegrade angles (subgroup B) and, in 10 patients, BT injection was combined with subsequent balloon dilatation (subgroup C). Immediate clinical response was achieved in 93% of patients. Clinical remission was sustained beyond 3 months in 83% of patients (responders). Fourteen responders (41%) did not experience a relapse during the median of 22 months. Twenty responders (59%) experienced symptomatic relapse approximately 8 months after the injection. Ten relapsers underwent BT reinjection, five (50%) of them were asymptomatic for another 14 months. The remaining five (50%) patients reported a second relapse approximately 6 months after the reinjection. Median duration of the symptom-free period was 11.5 months after the first BT injection, and 10.5 months after the second (P = 0.21). We did not find any significant predictor of a favorable outcome; responders tended to be older and to have a lower basal lower-esophageal-sphincter pressure. Patients in subgroup C were more likely to be in remission at 1 and 2 years as compared with patients in subgroup A. BT injection is an effective treatment of achalasia in the short term. However, almost 70% of patients experience a relapse within 2 years. BT injection should therefore be reserved for patients at risk for more invasive procedures or for patients who prefer this treatment.