Photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA-PDT) is an attractive alternative to PDT with porfimer sodium for the treatment of high-grade dysplasia (HGD) in Barrett's oesophagus (BO) because of the shorter duration of light photosensitivity and low risk of oesophageal stricture formation. Published results, however, show marked variation in its efficacy, and optimum treatment parameters have not been defined. This study investigated how the dose of ALA and the colour of the illuminating light influenced the biological effect. Twenty-seven patients were enrolled into a randomised controlled trial of red versus green (635 nm or 512 nm) laser light activation for the eradication of HGD with ALA-PDT in Barrett's oesophagus. A further 21 patients were subsequently treated with the most effective regimen. Regular endoscopic follow-up with quadrantic biopsies every 2 cm was performed. The primary outcome measure was eradication of HGD. Patient's receiving ALA at 30 mg/kg relapsed to HGD more than those receiving 60 mg/kg (P = 0.03). Additionally, for those treated with ALA 60 mg/kg, red laser light was more effective than green laser light (P = 0.008). Kaplan-Meier analysis of the 21 patients who were subsequently treated with this optimal regimen demonstrated an eradication rate of 89% for HGD and a cancer-free proportion of 96% at 36 months' follow-up. Using an ALA dose of 60 mg/kg activated by 1,000 J/cm red laser light, we found that ALA-PDT was a highly effective treatment for high-grade dysplasia in Barrett's oesophagus.