When the amino acid usage of all completely sequenced prokaryotes is studied by multivariate analysis (MVA), it is known that the genomic molar content of guanine plus cytosine (GC) and optimal growth temperature (Topt) have a dominant effect. Furthermore, these two factors are associated to the first two axes of different MVA, and thus, nearly independent among them. However, it was recently shown that for several Families of prokaryotes there are significant and positive correlations between GC and Topt. This trend is particularly clear within Bacillaceae, where there are species displaying a broad range of variations for these two factors. In this paper we report that (a) Topt and genomic GC are the main factors shaping amino acid usage but are not independent between them, (b) the usage of cysteine is the second source of variability, and finally (c) the global hydrophobicity of the encoded proteins of each species is the third main factor. (C) 2004 Elsevier Inc. All rights reserved.