In previous studies, bacterial cultures were isolated that had the ability to degrade the nitrate ester glyceryl trinitrate (i.e., nitroglycerin). The goal of the present study was to examine the ability of resting cells and cell-free extracts of the isolate Bacillus sp. ATCC 51912 to degrade the more recalcitrant nitrate ester propylene glycol dinitrate (PGDN). It was observed that the PGDN-denitrating activity was expressed during growth even when cells were cultured in the absence of nitrate esters. This indicates that nitrate esters are not required for expression of denitration activity. Using cell-free extracts, PGDN was observed to be sequentially denitrated to propylene glycol mononitrate (PGMN) and propylene glycol with the second denitration step proceeding more slowly than the first. Also it was observed that dialysis of the cell-free extracts did not affect denitration activity indicating that regenerable cofactors [e.g., NAD(P)H or ATP] are not required for denitration.