Previous studies have shown that Bacillus subtilis cells lacking penicillin-binding protein 1 (PBP1), encoded by ponA, have a reduced growth rate in a variety of growth media and are longer, thinner, and more bent than wild-type cells. It was also recently shown that cells lacking PBP1 require increased levels of divalent cations for growth and are either unable to grow or grow as filaments in media low in Mg2+, suggesting a possible involvement of PBP1 in septum formation under these conditions. Using epitope-tagging and immunofluorescence microscopy, we have now shown that PBP1 is localized at division sites in vegetative cells of B. subtilis. In addition, we have used fluorescence and electron microscopy to show that growing ponA mutant cells display a significant septation defect, and finally by immunofluorescence microscopy we have found that while FtsZ localizes normally in most ponA mutant cells, a significant proportion of ponA mutant cells display FtsZ rings with aberrant structure or improper localization, suggesting that lack of PBP1 affects FtsZ ring stability or assembly. These results provide strong evidence that PBP1 is localized to and has an important function in the division septum in B. subtilis. This is the first example of a high-molecular-weight class A PBP that is localized to the bacterial division septum.