A design-of-experiments approach was used to examine the effect of hexadecylpyridinium chloride (HPC), alone or in combination with the antibiotics vancomycin and natamycin, on the growth of Mycobacterium avium subsp. paratuberculosis (MAP). At concentrations above 74.4 microg/ml, HPC had a highly significant detrimental effect on the growth of MAP, whereas natamycin at 10.8 and 21.6 microg/ml and vancomycin at 5.2 and 10.4 microg/ml did not have such an effect. Titration of the amount of HPC tolerated by MAP indicated that growth can occur in the presence of 24.8 microg/ml or lower. Processing of bovine fecal specimens indicated that reducing the concentration of HPC from 32.22 to 1.07 mg/ml during decontamination may improve detection when cultures are grown on solid medium but not when cultures are grown in liquid medium. Further investigation into optimizing HPC concentration during processing of fecal samples is warranted. Natamycin, in conjunction with vancomycin, may be useful for controlling fungal contamination during isolation of MAP from fecal samples.