Bacitracins are a group of widely used peptide antibiotics. There has been interest in determining the three-dimensional structure of the bacitracins. However, solution studies indicate significant flexibility in their structure and to date native bacitracins have resisted attempts at crystallisation despite considerable efforts over a number of years by several groups. Here we report the first three-dimensional X-ray structure of a bacitracin, complexed to a subtilisin proteinase. X-Ray diffraction data were collected using synchrotron radiation in combination with the Image Plate Scanner system. The complex structure including two enzymes, two bacitracins, 220 water molecules and two Ca2+ ions was refined by restrained least-squares to a crystallographic R factor ( = sigma [[Fo-Fc]]/sigma [Fo]]) of 16.3% at 2.0 A.