We showed in a previous study that associations of antimicrobial peptides (AMPs), which are key components of the innate immune systems of all living species, with the fluoroquinolone enrofloxacin can successfully cure HeLa cell cultures of Mycoplasma fermentans and M. hyorhinis contamination. In the present work, the in vitro susceptibility of M. pulmonis, a murine pathogen, to enrofloxacin and four AMPs (alamethicin, globomycin, gramicidin S, and surfactin) was investigated, with special reference to synergistic associations and the effect of the mycoplasma cell concentration. Enrofloxacin and globomycin displayed the lowest MICs (0.4 microM), followed by gramicidin S (3.12 microM), alamethicin (6.25 microM), and surfactin (25 microM). When the mycoplasma cell concentration was varied from 10(4) to 10(8) CFU/ml, the MICs of enrofloxacin and globomycin increased while those of the three other molecules remained essentially constant. The minimal bactericidal concentration of enrofloxacin (0.8 microM) was also lower than those of the peptides (6.25 to 100 microM), but the latter killed the mycoplasma cells much faster than enrofloxacin (2 h versus 1 day). The use of the AMPs in association with enrofloxacin revealed synergistic effects with alamethicin and surfactin. Interestingly, the mycoplasma-killing activities of the two combinations enrofloxacin (MIC/2) plus alamethicin (MIC/4) and enrofloxacin (MIC/2) plus surfactin (MIC/16) were about 2 orders of magnitude higher than those of the three molecules used separately. These results support the interest devoted to AMPs as a novel class of antimicrobial agents and pinpoint their ability to potentiate the activities of conventional antibiotics, such as fluoroquinolones.