Pro-^K is the inactive precursor of ^K, a mother cell-specific sigma factor responsible for the transcription of late sporulation genes of Bacillus subtilis. Upon subcellular fractionation, the majority of the pro-^K was present in the membrane fraction. The rest of the pro-^K was in a large complex that did not contain RNA polymerase core subunits. In contrast, the majority of the ^K was associated with core RNA polymerase. Virtually identical fractionation properties were observed when pro-^E was analyzed. Pro-^K was completely solubilized from the membrane fraction and the large complex by Triton X-100 and was partially solubilized from the membrane fraction by NaCl and KSCN. The membrane association of pro-^K did not require spoIVF gene products, which appear to be located in the mother cell membrane that surrounds the forespore, and govern pro-^K processing in the mother cell. Furthermore, pro-^K associated with the membrane when overproduced in vegetative cells. Overproduction of pro-^K in sporulating cells resulted in more pro-^K in the membrane fraction. In agreement with the results of cell fractionation experiments, immunofluorescence microscopy showed that pro-^K was localized to the mother cell membranes that surround the mother cell and the forespore in sporulating wild-type cells and mutant cells that do not process pro-^K. Treatment of extracts with 0.6 M KCl appeared to free most of the pro-^K and ^K from other cell constituents. After salt removal, ^K, but not pro-^K, reassociated with exogenous core RNA polymerase to form holoenzyme. These results suggest that the prosequence inhibits RNA polymerase core binding and targets pro-^K to the membrane, where it may interact with the processing machinery.