Prebiotics are non-digestible food ingredients that target selected groups of the human colonic microflora, thus having the ability to alter the composition towards a more 'beneficial' community, i.e. selectively increasing populations of bifidobacteria and/or lactobacilli. In the present study the prebiotic potential of partially hydrolysed guar gum (PHGG) and fructo-oligosaccharides (FOS) in a biscuit was assessed in human volunteers. Fluorescent in situ hybridization using oligonucleotide probes targeting Bacteroides spp., Bifidobacterium spp., Clostridium spp. and Lactobacillus-Enterococcus spp. were used for the bacteriology and total bacteria were enumerated using the fluorescent stain 4',6-diamidino-2-phenylindole. Thirty-one volunteers consumed daily either three experimental biscuits (providing a total (g/d) of 6.6 FOS and 3.4 PHGG) or three placebo biscuits for two 21-d crossover periods. Bifidobacteria significantly increased in number on ingestion of the experimental biscuits compared with pre-treatment and placebo population levels. Bifidobacterial numbers returned to pretreatment levels within 7 d of the cessation of intake of experimental biscuits. A correlation was observed between the initial faecal bifidobacterial numbers and the magnitude of bifidogenesis, with volunteers who possessed low initial population levels of bifidobacteria experiencing the greatest increase in bifidogenesis. No changes were observed in the other bacterial groups monitored during the trial. Thus, the prebiotic nature of FOS and PHGG was maintained in a final food product as evidenced from the selective increase in bifidobacterial numbers.