A genome and fatty acid analysis of 16 Pseudomonas stutzeri reference strains having DNA compositions ranging from 62.2 to 65.5 mol% G+C was performed by pulsed-field gel electrophoresis of XbaI and SpeI macrorestriction fragments and gas chromatography of total cellular fatty acids. Macrorestriction fragment patterns were evaluated by using previously described algorithms (D. Grothues and B. Tümmler, Mol. Microbiol. 5:2763-2776, 1991), and the results allowed us to subdivide the species into two groups which correlated with G+C content. Two examples of recent strain divergence were observed among clinical isolates, but in general a marked degree of heterogeneity was observed in the macrorestriction fragment patterns, and even phenotypically similar strains produced divergent patterns. While the differences were not sufficiently great to exclude any strain from P. stutzeri, they suggest that recombination and niche-specific selection may be significant factors responsible for generating and maintaining the heterogeneity inherent in the species. Genome sizes were estimated from the sums of SpeI restriction fragment sizes and ranged from 3.4 to 4.3 Mbp; the genome sizes of the low-G+C-content strains (G+C contents, approximately 62 mol%) were confined to a narrow range between 3.9 and 4.1 Mbp. An examination of the distributions of macrorestriction fragments resulting from digestion with XbaI and SpeI showed that both distributions differed significantly from the expected (random) distribution, suggesting that there is a supragenic level of chromosomal organization. An analysis of fatty acid methyl ester data by using Microbial Identification System software revealed a similar correlation between phenotype and G+C content, indicating that division of the species is possible by the method used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)