A positive regulatory gene (preL) from Lactobacillus sp. no. 1, which enhanced production of neutral protease (NprL), was cloned in Bacillus subtilis DB104 together with the nprL structural gene. When nprL was expressed with the regulatory gene (preL) on the same plasmid, extracellular protease production in a 12 h culture was 28-fold higher (14.2 U/ml) than protease production without preL expression (0.5 U/ml). The nucleotide sequence of the preL was determined and the deduced amino acid sequence revealed that PreL was relatively homologous (33%) to that of a positive regulatory protein (NprA) for protease production from Bacillus stearothermophilus, whose function is similar to that of preL. Northern hybridization analysis revealed that preL, in multicopy, enhanced transcription of the nprL gene. A consensus DNA binding motif (helix-turn-helix) was found within the amino acid sequence of the PreL. These results suggest that preL is a transcriptional activator of nprL. Indeed, a possible target sequence of PreL with a stable palindromic structure containing putative -10 region was found in the promoter region of nprL. It is speculated that PreL melts this palindromic structure and assists RNA polymerase in binding effectively to the nprL promoter. Furthermore, a similar palindromic structure was also found in the promoter region of preL itself. Enhanced production of NprL in the stationary phase may possibly be due to this auto-activation by preL.