A study was conducted with conventional and germ-free broiler chicks to obtain more information on the role of the intestinal microflora in the anti-nutritive effects of NSP in broiler chicks. As the NSP source, highly methylated citrus pectin (HMC) was used at a dose level of 30 g/kg in a maize-based diet. The diets fed to the germ-free chicks were gamma-irradiated, whereas those fed to the conventional chicks were not. Feeding the HMC diet to conventional birds depressed weight gain and food utilization (P < 0.05), whereas in germ-free birds only weight gain was reduced (P < 0.05). Feeding the HMC diet to conventional birds reduced digestibilities of energy and starch at the end of the jejunum. Ileal digestibilities of starch and energy were not strongly affected when birds were fed on the HMC-containing diet. Faecal digestibilities of organic matter, crude fat, starch and amino acids, N retention and metabolizable energy were reduced when conventional chicks were fed on the HMC diet. Feeding the HMC diet to germ-free birds hardly affected faecal digestibility of nutrients and N retention, whereas metabolizable energy was increased. Feeding the HMC diet to conventional or germ-free birds increased the viscosity of the digesta in the small intestine. This increase in digesta viscosity was more pronounced in conventional than in germ-free birds. The pH of ileal digesta was reduced when HMC was added to the diet of conventional chicks, but not in germ-free chicks. Feeding the HMC diet to conventional birds markedly affected morphology of the gut wall, whereas in germ-free chicks very little effect was found on gut morphology. Based on the results of the present study, it is concluded that the gastrointestinal microflora mediates the magnitude of the anti-nutritive effects of HMC in broiler chicks. However, the exact role of the microflora in chicks in the magnitude of the anti-nutritional effects of HMC could not be derived from the present study, since the results might have been influenced by gamma-irradiation of the diets fed to the germ-free chicks.