Plasmid pUB110, isolated from vegetative cells of Bacillus subtilis, has an average of 34 negative supertwists (tau av = -34). This value falls to -30 early in sporulation, and the plasmid in the mother cell compartment maintains a tau av of -30. However, the plasmid within the developing forespore becomes much more negatively supercoiled, reaching a tau av of -47 in the dormant spore. This increased negative supercoiling in the forespore plasmid takes place in parallel with the synthesis of small, acid-soluble spore proteins, alpha and beta; and the plasmid from spores lacking small, acid-soluble proteins alpha and beta has a tau av of -40. The large increase in negative supercoiling of spore plasmid was also observed with Bacillus megaterium and in B. subtilis containing a plasmid with an origin different from that of pUB110. During spore germination plasmid pUB110 rapidly relaxed back to the tau av value characteristic of vegetative cells. It is possible that the observed changes in forespore plasmid topology are involved in modulating gene expression, DNA photochemistry, or both of these parameters in this compartment.