The Bacillus subtilis rpsD gene, which encodes ribosomal protein S4, is subject to autogenous regulation. Repression of rpsD expression by excess S4 protein was previously shown to be affected by mutations in the leader region of the gene. A large number of deletion and point mutations in the leader region were generated, and their effect on repression by S4 in vivo was tested. These studies indicated that the required region was within positions +30 to +190 relative to the transcription start point. Replacement of the rpsD promoter with a lac promoter derivative which is expressed in B. subtilis had no effect, indicating that repression by S4 occurs at a level subsequent to transcription initiation. The rpsD leader region was isolated from several Bacillus species. Members of the B. subtilis group, as defined by analysis of 16S rRNA sequence, contained a leader region target site very closely related in structure to that of B. subtilis, despite considerable primary sequence variation; the B. brevis rpsD leader contained some but not all of the structural features found in the regulatory target sites of the other Bacillus species. Very little similarity to the Escherichia coli alpha operon S4 target site was found at either the primary-sequence or the secondary-structure level. Mutagenic and phylogenetic data indicate that the secondary structure of the leader region regulatory target site contains two large stem-loop domains. The first of these helices has a side loop which is essential for autoregulation, is highly conserved among Bacillus rpsD genes, and is similar to a region of 16S rRNA important in S4 binding.