Under certain growth conditions, Bacillus subtilis can develop natural competence, the state in which it is able to bind, adsorb and incorporate exogenous DNA. Development of competence is a bistable process and is subject to complex regulation. Rok is a repressor of the key transcriptional activator of competence genes, comK, and limits the size of the subpopulation that develops competence. Here we report the finding that the large conjugative B. subtilis plasmid pLS20 harbours a rok homologue rokLS20. Although the deduced product of rokLS20 is considerably shorter than the chromosomally encoded Rok protein, we show that ectopic expression of the plasmid-encoded RokLS20 leads to inhibition of competence by repressing comK, and that the effects of the plasmid and chromosomally encoded Rok proteins are additive. We also show that pLS20 inhibits competence in a rokLS20-dependent manner and that purified RokLS20 preferentially binds to the comK promoter. By analysing the available databases we identified several additional rok-like genes. These putative rok genes can be divided into two groups and we propose that rokLS20 is the prototype of a newly identified subgroup of nine rok genes. Finally, we discuss the possible role of the plasmid-located rok and its relatedness with other rok genes.