Signal peptides are essential N-terminal extensions in export proteins, and have a positively charged N-terminus, a hydrophobic central core, and a C-terminal cleavage region. They interact in a consecutive manner with different accessory proteins during the secretion process. Potential patterns or periodicity in the amino acid (aa) sequence were searched, using multivariate techniques, for a large number of signal peptides from mollicutes (mycoplasmas), other Gram-positive bacteria, and Escherichia coli. Mollicutes signal peptides were significantly different from the E. coli and Gram-positive ones by their N-terminal charge, peptide length, and especially, unique periodicities of side chain hydrophobicity and volumes. Their lipoprotein signal peptides were longer than for any other bacteria. Significant differences were also recorded between the other bacterial peptide groups. Specific aa patterns were more related within the signal peptides from several groups of secreted bacillus enzymes, than for all signal peptides from one bacillus species. In E. coli, signal peptides from proteins routed for the various destinations revealed significant and compartment-specific sequence patterns not evident by other methods. This was substantiated from a large number of signal peptide secretion mutants for the E. coli periplasmic space. It is proposed that the differences in aa patterns and side-chain properties are related to the secondary structure sidedness and topology of the signal peptides, and important for specific interactions during the secretion process.