The occurrence ofAcetobacter diazotrophicuswas directly demonstrated in plant tissues using a species-specific oligonucleotide probe and polymerase chain reaction (PCR) amplification of a 411-bp product. The oligonucleotide probe was derived from the sequence of a highly variable region of 23S rDNA and its specificity was tested with membrane-bound nucleic acids of 112 different microorganisms in hybridization experiments. It was found to be able to discriminateAcetobacter diazotrophicusfrom otherAcetobacterspp. and other reference organisms. PCR amplification from pure cultured cells or colonies showed that the method was sensitive enough to detect as few as 200 cells in the reaction. The presence ofAcetobacter diazotrophicusin tissues of micropropagated sugarcane plants inoculated with either this bacterium or a mixture of this bacterium andHerbaspirillum seropedicaewas demonstrated by PCR amplification.Acetobacter diazotrophicuscould also be detected by the PCR method in field-grown sugarcane plants, as well as in certain cultivars ofPennisetum purpureumSchumach but not in\i maize, sweet potato, and two samples of weed plants grown within or outside of a sugarcane field.The addition of 1% polyvinylpolypyrrolidone during preparation of the field samples, especially with root tissues, improved the amplificability of the target sequence. The minimum level of detection of this bacterium in sugarcane tissue using the universal 1440 and AD species-specific primers was about 105bacterial cells/g of fresh plant material. The sensitivity could be improved 10-fold by probing immobilized PCR products containing the target region with the32P-labeled oligonucleotide AD.Key words:Acetobacter diazotrophicus, diazotrophic endophytes, specific rRNA-targeting oligonucleotides, polymerase chain reaction (PCR).