BACKGROUND Levator ani syndrome is characterized by anorectal discomfort/pain, treatment of which is unsatisfactory. We hypothesized that Botulinum toxin relieves spasm and improves symptoms. OBJECTIVE To perform a randomized, placebo-controlled, crossover study to examine the efficacy and safety of botulinum toxin in patients with levator ani syndrome. METHODS Twelve patients with levator ani syndrome (>or=1 year) received anal intra sphincteric injections of 100 units of botulinum toxin A and placebo at 90-day intervals using EMG guidance. Daily frequency, severity, duration and intensity of pain (VAS) were recorded. Anorectal manometry, balloon expulsion and pudendal nerve latency tests were performed to examine the physiological changes and adverse effects. RESULTS Seven patients (male/female = 4/3) completed the study and three had incomplete data, but all 10 underwent in an ITT analysis; two others dropped out. After administration of botulinum toxin, the mean frequency, intensity and duration of pain were unchanged (P = 0.31) compared with baseline. The 90-day mean VAS pain score was 6.79 +/- 0.27 vs. baseline score of 7.08 +/- 0.29 (P = 0.25). Anal sphincter pressures, rectal sensory thresholds, pudendal nerve latency and balloon expulsion times were unchanged after drug or placebo administration. CONCLUSIONS Injection of botulinum toxin into anal sphincter is safe, but it does not improve anorectal pain in levator ani syndrome.