Sequencing of a 1.3-kb fragment of DNA from the temperate Lactococceus lactis subsp. cremoris phage phiLC3 revealed a pair of two divergently oriented ORFs, orf63 and orf286. The deduced amino acid sequence of the product of orf286 showed extensive homology to those of repressors of the temperate lactococcal phages rlt, Tuc2009 and BK5-T. A mutant with an amber mutation in orf286 gave rise to a clear plaque phenotype, indicating that this gene is involved in the lytic and lysogenic development of phiLC3. Gel mobility shift assays showed that the partially purified Orf286 protein bound specifically to the 224-bp intergenic region located between orf286 and orf63, and further characterization by DNase I footprinting analysis revealed that Orf286 protects two distinct sites within this region. Sequence analysis of the intergenic region revealed two putative, divergently oriented promoters, P1 and P2; orf286 and orf63 are probably transcribed from P1 and P2, respectively. In vivo analyses of P1 and P2 using beta-galactosidase as a reporter enzyme in L. lactis showed that transcription from P1 was repressed while transcription from P2 was stimulated in the presence of the Orf286 protein. These results suggest a complex role for the Orf286 protein in regulating the genetic switch between lytic and lysogenic growth of phiLC3.