Aims: The isolation of high-quality RNA from cheese is a prerequisite for analysis of in situ gene expression of dairy micro-organisms. Methods and Results: A method for rapid isolation of bacterial cells from cheese using cold citrate buffer followed by mechanical cell disruption was developed. RNA was extracted from experimental ultrafiltration (UF) cheeses (at 2, 8, 24 h, 7 and 14 days) and from Cheddar cheese (from 1 day to 1 year). The quantity and quality of the extracted RNA was assessed. The transcript abundance of seven genes (tuf, gapB, purM, cysK, ldh, cit and gyrA) was estimated by reverse transcription real-time PCR. In UF cheeses, the quantity of RNA extracted increased from 0.2 to 24 mu g g(-1), with an RNA Integrity Number (RIN) above 9. In the experimental Cheddar cheeses, the RNA extraction yield decreased from 67.7 mu g g(-1) after 1 day to 23.7 mu g g(-1) after 6 months, with RIN value above 9 during the first month. The transcript abundance of the seven genes demonstrated metabolic activity of lactococci after several weeks of ripening in both cheeses. Significance and Impact of the Study: The method described produced large quantities of high-quality RNA for future whole genome expression studies in cheese.