The effectiveness of long-term treatment of Alzheimer's disease with cholinesterase inhibitors is a matter of controversy. We evaluated the effects of prolonged treatment with eptastigmine in 176 patients with mild to moderate Alzheimer's disease participating in the open-label extension phase of a 25-week double-blind, placebo-controlled trial of eptastigmine. The effects of eptastigmine on cognition and daily functioning were evaluated with the cognitive portion of the Alzheimer's Disease Assessment Scale (ADAS-Cog) and the Instrumental Activities of Daily Living (IADL) scale, respectively. Safety was monitored by physical examination, laboratory tests, vital functions and electrocardiogram measurements and by the assessment of adverse events. One hundred and fifty-three patients (87%) completed 1 year of treatment, 77 patients (44%) 18 months and 33 patients (19%) 2 years of treatment. Patients treated for 2 years showed an improvement of mean ADAS-Cog scores compared to baseline for 31 weeks and mean IADL scores remained close to baseline for 25 weeks. Cognitive and functional scores then worsened as expected in this progressive disease. After 2 years, patients deteriorated compared to baseline by 13.4 points on the ADAS-Cog and 6.1 points on IADL. Historical untreated controls with identical disease severity are expected to have an annual worsening of approximately 10.9 points on ADAS-Cog and 4.9 points on IADL. Thus patients treated with eptastigmine for 2 years had a benefit of 8.5 points on ADAS-Cog and 3.8 points on IADL. These benefits translate to about 9 months difference between eptastigmine-treated patients and untreated historical patients. The drug was generally well tolerated with 14 patients (7.9%) withdrawing due to adverse events. Adverse events, not necessarily drug-related, were recorded in 66 patients (37.5%) and were transient and generally mild in severity. This study indicates that prolonged treatment with eptastigmine is safe and produced a clinically long-term benefit in patients with Alzheimer's disease.