Lactococcus lactis subsp. cremoris is of considerable interest to the dairy industry, which relies upon the few available strains for the manufacture of cheddar cheese free of fermented and fruity flavors. The subspecies cremoris differs from related subspecies by the lack of a few phenotypic traits. Our purpose was to identify unique rRNA sequences that could be used to discriminate L. lactis subsp. cremoris from related subspecies. The 16S rRNAs from 13 Lactococcus strains were partially sequenced by using reverse transcriptase to identify domains unique to L. lactis subsp. cremoris. All five strains of the subspecies cremoris had a unique base sequence in a hypervariable region located 70 to 100 bases from the 5' terminus. In this region, all L. lactis subsp. lactis biovar diacetylactis strains examined had a sequence identical to that of L. lactis subsp. lactis 7962, which was different from other strains of the subspecies lactis by only one nucleotide at position 90 (Escherichia coli 16S rRNA structural model) (J. Brosius, J. L. Palmer, J. P. Kennedy, and H. F. Noller, Proc. Natl. Acad. Sci. USA 75:4801-4805, 1978). Oligonucleotide probes specific for the genus Lactococcus (212RLa) and for the subspecies cremoris (68RCa) were synthesized and evaluated by hybridization to known rRNAs as well as fixed whole cells. Efficient and specific hybridization to the genus-specific probe was observed for the 13 Lactococcus strains tested. No hybridization was seen with the control species. All five strains of the subspecies cremoris hybridized to the subspecies-specific probe.