Protamines are cationic peptides that exert antimicrobial activity. We have examined the evolution of bacterial resistance to protamine sulphate and the resulting effects on fitness and physiology, with the objective of increasing knowledge about mechanisms of bacterial resistance to antimicrobial peptides. Spontaneous, protamine-resistant Salmonella enterica serovar Typhimurium (i.e. Salmonella Typhimurium) LT2 mutants were isolated on agarose plates containing protamine sulphate. Resistance mutations were identified using transposon insertions and DNA sequencing. Peptide susceptibility was determined by broth dilution tests and antibiotic susceptibility using Etests. Fitness was determined as log-phase growth rates. Growth-compensated strains were isolated by serial passage through population bottlenecks followed by visual screening for large colonies. Protamine-resistant mutants appeared at a rate of 2.3 x 10(-7)/cell/generation. These mutants were 2-20 times more resistant to protamine than the parental strain and less susceptible to several other antimicrobials, including colistin, gentamicin, lactoferricin and human defensin HNP-1. The resistance mutations were mapped to genes involved in haem biosynthesis and respiration, and were associated with a reduction in bacterial fitness. Some mutants could, in the absence of protamine, be evolved to higher fitness by acquiring second-site compensatory mutations. Spontaneous mutants resistant to protamine sulphate were readily selected in Salmonella Typhimurium LT2. These mutants were less susceptible to several other peptides and antibiotics, and had the characteristics of small colony variants, a phenotype often associated with persistent and recurrent infections that are difficult to treat and which could be a strategy for bacteria to escape the killing effects of antimicrobial peptides.