To characterize the antimicrobial protein produced by Bacillus coagulans used in the probiotic dietary supplement (Lactospore((R)) Probiotic, Sabinsa Corp., Piscataway, NJ, USA). Bacillus coagulans ATCC 7050 was grown at 37 degrees C for 18 h. The cell free supernatant was concentrated 10-fold (lactosporin preparation, LP). The antimicrobial activity of LP was confirmed against Micrococcus luteus ATCC 10420 in a well diffusion assay. The proteinaceous nature of LP was determined following exposure to different enzymes. The activity of LP was pH-dependent but stable to heat. The isoelectric point of LP was determined to be 3.5-4.0. PCR analyses showed no similarity between lactosporin and known antimicrobial proteins produced by the Bacillus spp. Lactosporin is a novel antimicrobial protein. Initial characterization indicates that it may fall outside of the conventional classification of class I and II bacteriocins. Loss of activity after exposure to a number of proteolytic enzymes and lipase suggest that lactosporin may posses a lipid moiety which contributes to its inhibitory activity. The unique characteristics of lactosporin, including its antimicrobial activity against pathogenic micro-organisms, indicate that it may have potential for application in foods and personal care products.