Spo0A is a two-domain response regulator required for sporulation initiation in Bacillus subtilis. Studies on response regulators have focused on the activity of each domain, but very little is known about the mechanism by which the regulatory domain inhibits the activator domain. In this study, we created a single amino acid substitution in the regulatory domain, D75S, which resulted in a dramatic decrease in sporulation in vivo. In vitro studies with the purified Spo0AD75S protein demonstrated that phosphorylation and DNA binding were comparable with wild type Spo0A. However, the mutant was unable to stimulate transcription by final sigma(A)-RNA polymerase from the Spo0A-dependent spoIIG operon promoter. We suggest that the amino acid Asp(75) and/or the region within which it resides, the alpha3-beta4 loop, are involved in the inhibitory interaction between the regulatory and activator domains of Spo0A.