Each prokaryote has a unique genomic signature as evidenced by a set of species-specific frequencies of short oligonucleotides. With respect to genomic signatures a bacterial genome is homogenous and the variation within a genome is smaller than the variations between genomes of different species. This study quantifies the species-specificity of genomic signatures in the complete genomes of 57 prokaryotes. The species-specificity in the genomic signature was related to the quantification of other sequence biases, such as G + C content, synonymous codon choice and amino acid usage. The results confirm that the genomic signature is genome-wide with high species-specificity in both coding and non-coding regions. In coding regions the species-specific bias in synonymous codon choice was comparable to the genomic signature, while the bias in amino acid usage only captured about 50% of the species-specific bias in the genomic signature. A correlation between the species-specificity in synonymous codon choice and amino acid usage was identified, in which proteins with species-specific amino acid usage were also coded with species-specific synonymous codon choice. However, we demonstrated that the G + C content captures only approximately 40% of the species-specificity in the genomic signature, and is insufficient to explain the species specificity in the non-coding regions. Thus, the species-specific bias in non-coding regions remains largely unknown. Further, we compared the genomic signature in relation to phylogenetic distance. This was performed in order to illustrate the feasibility of a hierarchical classification scheme in future applications of the described classification methodology in screening for horizontal gene transfer and biodiversity studies. (C) 2003 Elsevier Science B.V. All rights reserved.