An oligonucleotide-microarray method was developed for the detection of intestinal bacteria in fecal samples collected from human subjects. The 16S rDNA sequences of 20 predominant human intestinal bacterial species were used to design oligonucleotide probes. Three 40-mer oligonucleotides specific for each bacterial species (total 60 probes) were synthesized and applied to glass slides. Cyanine5 (CY5)-labeled 16S rDNAs were amplified by polymerase chain reaction (PCR) from human fecal samples or bacterial DNA using two universal primers and were hybridized to the oligo-microarray. The 20 intestinal bacterial species tested were Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides fragilis, Bacteroides distasonis, Clostridium clostridiiforme, Clostridium leptum, Fusobacterium prausnitzii, Peptostreptococcus productus, Ruminococcus obeum, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus albus, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium infantis, Eubacterium biforme, Eubacterium aerofaciens, Lactobacillus acidophilus, Escherichia coli, and Enterococcus faecium. The two universal primers were able to amplify full size 16S rDNA from all of the 20 bacterial species tested. The hybridization results indicated that the oligo-microarray method developed in this study is a reliable method for the detection of predominant human intestinal bacteria in the fecal samples.