Reverse transcriptase sequence analyses of variable regions of 16S rRNA of the nitrogen-fixing (Nif+)Frankia strain Ag45/Mut 15 and the Nif− strains AgB1.9 and AgW1.1 showed large differences in two of three variable regions between bothFrankia groups. Synthetic oligonucleotides complementary to sequences in one of these different regions were used in hybridization experiments against isolated rRNA of severalFrankia strains belonging to three compatibility groups. Ribosomal RNA of eleven effectiveFrankia strains obtained from differentAlnus species strongly hybridized with the probe against the effective strain Ag45/Mut 15 (probe EFP), whereas ineffective strains and effective strains obtained from other hosts (Elaeagnus, Comptonia, Coriaria, Hippophae, Colletia spp.) did not hybridize. Strong hybridization was also obtained with the effectiveCasuarina strain CcI3. In the group of effective alder strains one strain showed weaker hybridization indicating small sequence differences. Different sequences were also found after hybridization with the probe against the ineffectiveFrankia strains AgB1.9 and AgW1.1 (probe IFP). Only these two strains showed hybridization. The same results were obtained byin-situ hybridizations with probe EFP, whereas hybridization with probe IFP showed crossreaction with several other strains. Tests of these probes against rRNA of several microorganisms indicate a high specificity.