We have investigated the value of rRNA gene restriction (rDNA) patterns for strain discrimination in aeromonads isolated from clinical and environmental sources. Chromosomal DNA was purified and digested with restriction enzymes Pst I and Sma I, and the resulting fragments were separated by agarose gel electrophoresis and stained with ethidium bromide. The DNA was transferred to membranes (Southern blotting), where it was probed with biotinylated plasmid pKK3535 DNA. Hybridization bands were developed with the BluGene (BRL) kit. The resulting rDNA patterns were visually analyzed for similarities between epidemiologically related strains. Pattern heterogeneity was observed among unrelated strains of aeromonads of clinical and environmental origin, whereas homology was observed among Aeromonas caviae isolates from three siblings with gastroenteritis and between an infant and foster mother with acute gastroenteritis caused by A. caviae. Multiple isolates of A. caviae from the same patient over more than 3 years produced homologous rDNA patterns which remained stable after 20 consecutive subcultures. Serotyping results for typable strains were consistent with strain designations derived by analysis of rDNA patterns. The specificity, reproducibility, stability, and ease of performance, especially with the nonisotopic probe, make ribotyping a valuable method for epidemiological investigations of Aeromonas infections.