3H-labeled rRNA was prepared from Acinetobacter calcoaceticus ATCC 23055Tand hybridized with filter-fixed DNA from carefully selected Acinetobacter strains, reference strains of taxonomically well-located genera, or some selected misnamed strains. The hybridization parameters, the temperature at which 50% of the hybrid was denatured (Tm(e)) and percent rRNA binding, were determined. The following conclusions can be drawn from the DNA:rRNA hybridization results, the rRNA similarity map, and a Tm(e) dendrogram. (i) The genus Acinetobacter is rather heterogeneous. It covers a 5°C Tm(e) range. (ii) A combination of our hybridization data and data from the literature indicates that several additional strains belong in the genus Acinetobacter. (iii) Acinetobacter Iwoffii ATCC 17960 is certainly not a member of the genus Acinetobacter. (iv) The genus Acinetobacter is not related to one of the well-established genera or families, nor is it a true member of one of the previously described rRNA superfamilies. The genus Acinetobacter forms a new and separate rRNA branch which is taxonomically slightly more close to rRNA superfamily II (Pseudomonas fluorescens complex, Azomonas, Azotobacter, etc.) than to rRNA superfamily I (Enterobacteriaceae, Vibrionaceae, Pasteurellaceae, etc.) and the genus Xanthomonas. (v) The misnamed species “Pseudomonas pavonacea” and “Pseudomonas cruciviae” belong to the Acinetobacter rRNA branch, although they differ from the genus Acinetobacter in several important phenotypic features.