Background: A prior Phase 3 study of sustained-release fampridine (4-aminopyridine) in MS showed beneficial effects on ambulation and leg strength. Objective: To confirm the efficacy and safety of fampridine (Fampridine-SR) in an independent study of patients with ambulatory deficits due to multiple sclerosis (MS). Methods: This was a randomized, double-blind, placebo-controlled, parallel-group study comparing 10 mg sustained-release fampridine bid and placebo. A 2-week placebo run-in was followed by 9 week treatment and 2 week follow-up periods. Eligibility criteria included: definite MS of any subtype; age 18-70; completion of the Timed 25-Foot Walk (T25FW) within 8-45 seconds at screening; stable concomitant medications. The pre-specified primary outcome was the proportion of patients with consistent improvement in walking speed on the T25FW during the treatment period (Timed Walk Responders). The clinical validity of the response criterion was previously established by correlation with the 12-Item MS Walking Scale, subject and clinician global impression scales. The secondary outcome was manually assessed leg strength, comparing Timed Walk Responders and Non-Responders with placebo-treated patients. Results: A total of 239 patients were randomized; 120 received fampridine and 119 placebo. 227 patients completed the trial (n = 113, 114). The fampridine-treated group had a higher proportion of Timed Walk Responders, compared to the placebo group (42.9 % v. 9.3 %; p<0.001) across all MS subtypes. Walking speed in Timed Walk Responders improved by approximately 25% from baseline, throughout the treatment period. Leg strength was significantly improved in Timed Walk Responders versus placebo-treated patients (p = 0.028). Adverse events were similar to those observed in previous studies of fampridine in MS. Three serious adverse events led to discontinuation, only one of which (patellar fracture) was in the fampridine group. Conclusions: Fampridine consistently improved walking speed and leg strength in a significant proportion of MS patients during 9 weeks of treatment.