Chromosomal DNA from 26 strains of Providencia stuartii isolated mainly in hospitals in the United Kingdom and reference strains of P. stuartii, P. rustigianii, and Proteus vulgaris were digested with the restriction endonucleases EcoRI and HindIII. After electrophoresis in agarose gels, the fragments were subjected to Southern blot hybridization analysis with a biotin-labeled cDNA probe transcribed from a mixture of 16S and 23S rRNA from P. stuartii NCTC 11800T. The pattern of bands (the rDNA fingerprint), which depended on restriction fragment length polymorphism containing rRNA genes, was used as a measure of minor genomic variation within and between species. The P. stuartii clinical isolates had similar total digest patterns, but the rDNA fingerprints revealed some heterogeneity between strains, with EcoRI digests providing better strain discrimination than HindIII. Such rDNA fingerprints comprised between five and seven bands with sizes in the range of 5 to 28 kilobases. The 11 different EcoRI patterns were compared by numerical analysis, and several groups or subgroups of strains were identified. Over half (15 of 26) of the urease-negative isolates (subgroups Aa and Ab) had patterns that differed only by the presence or absence of a 25-kilobase band. Urease-negative strains from other clinical material were more heterogeneous in their patterns. No correlation was apparent between strain pattern group and urease production or geographic location of isolate. The P. stuartii rDNA fingerprints were quite distinct from those of allied Providencia and Proteus species and provided a more sensitive measure of minor genomic differences than total DNA digests did.