This study was designed to assess the potential toxicity of pullulan, a starch-like substance produced by Aureobasidium pullulans that is proposed for use as a texturizer for meat and meat substitutes and as a flavour substrate. Sprague-Dawley rats (15/sex/group) were administered pullulan as a dietary admixture at levels of 1, 5 and 10% for a period of 62 wk. Control animals (15/sex) received untreated standard laboratory diet. The feeding study, originally intended to continue for 24 months, was terminated at 62 wk due to poor survival resulting from intercurrent pneumonia which was confirmed by histological findings. At 62 wk of treatment, all survivors were killed, complete gross post-mortem examinations were conducted on all animals, selected organs were weighed and organ/body weight and organ/brain weight ratios calculated. Mean body weights of all treated groups were comparable to controls. There were no indications of an adverse effect of pullulan on food consumption or food efficiency. At termination of the study, haematology and clinical chemistry values of treated animals were comparable to control values. There was no indication of pullulan-related toxicity in terminal organ and body weights. Macroscopic and microscopic examinations revealed no toxic lesions due to treatment. The mutagenicity of pullulan was assessed with and without metabolic activation in Salmonella typhimurium (TA100, TA1535, TA98 and TA1537). Pullulan did not increase the number of revertants per plate in any strain at any dose, including 10,000 micrograms/plate with or without metabolic activation, suggesting that it lacks mutagenic/carcinogenic potential on the basis of these results, it is concluded that pullulan lacks significant toxicological activity. The no-observed-adverse-effect level was 10% (equal to or greater than 4450 mg/kg body weight/day in males and 5080 mg/kg body weight/day in females) which would support use in various foods as a substrate for flavours.