The plasmid encoded LlaI R/M system from Lactococcus lactis ssp. lactis consists of a bidomain methylase, with close evolutionary ties to type IIS methylases, and a trisubunit restriction complex. Both the methylase and restriction subunits are encoded on a polycistronic 6.9 kb operon. In this study, the 5' end of the llaI 6.9 kb transcript was determined by primer extension analysis to be 254 bp upstream from the first R/M gene on the operon, llaIM. Deletion of this promoter region abolished LlaI restriction in L. lactis. Analysis of the intervening sequence revealed a 72-amino-acid open reading frame, designated IlaIC, with a conserved ribosome binding site and helix-turn-helix domain. Overexpression of llaIC in Escherichia coli with a T7 expression vector produced the predicted protein of 8.2 kDa. Mutation and in trans complementation analyses indicated that C-LlaI positively enhanced LlaI restriction activity in vivo. Northern analysis and transcriptional fusions of the llaI promoter to a lacZ reporter gene indicated that C-LlaI did not enhance transcription of the IlaI operon. Databank searches with the deduced protein sequence for IlalC revealed significant homologies to the E. coli Rop regulatory and mRNA stabilizer protein. Investigation of the effect of C-LlaI on enhancement of LlaI restriction in L. lactis revealed that growth at elevated temperatures (40°C) completely abolished any enhancement of restriction activity. These data provide molecular evidence for a mechanism on how the expression of a restriction system in a prokaryote can be drastically reduced during elevated growth temperatures, by a small regulatory protein.