pPL608-TR1 is a high-copy plasmid that permits phenotypic expression of the mouse dihydrofolate reductase (DHFR) gene in Bacillus subtilis. A plasmid mutation has been identified that increases expression of mouse DHFR more than ten-fold. The mutation is located in a 0.2-kb segment that intervenes between the DHFR gene and the 0.3-kb promoter fragment needed for transcriptional activation of DHFR. Nucleotide sequence analysis suggests that the effect of the mutation is to facilitate translation, initiated within the promoter fragment, through the 0.2-kb segment to the site of insertion of the DHFR-containing fragment. Additional promoter-containing fragments selected by their ability to promote expression of a plasmid gene located downstream from DHFR, the CAT gene, promote either high, intermediate or no phenotypic expression of DHFR. The results indicate that promoter fragments that allow phenotypic expression of the mouse DHFR gene contain two regulatory signals. One signal is essential to transcription of both DHFR and CAT and therefore functions as a promoter. The second signal may be necessary for translation of that portion of the mRNA specifying mouse DHFR.