The growth and activity of two Lactococcus strains and one Leuconostoc strain in CO2-acidified and non-acidified refrigerated milk were evaluated separately and as a mixed culture to determine their suitability for use as a starter in the manufacture of Afuega’l Pitu, an acid-coagulated Spanish cheese, from refrigerated CO2-acidified milk. The growth of the strains studied and their production of organic acids were similar in CO2-acidified and non-acidified refrigerated or fresh milk, indicating that CO2 treatment does not affect the metabolic activity of the strains. However, refrigeration enhanced the production of acetaldehyde, ethanol and diacetyl in CO2-acidified and non-acidified milk. The level of diacetyl was also greater in refrigerated CO2-acidified milk than in refrigerated non-acidified milk. It was concluded that refrigerated milk acidified with CO2 can be satisfactorily used in the manufacture of Afuega’l Pitu cheese, and that this technique can be also used in the production of other acid-coagulated cheeses.This work was financially supported by the Comisión Interministerial de Ciencia y Tecnología of Spain (Grant ALI96-0406). P. Ruas-Madiedo was the recipient of a fellowship from the Council of Villaviciosa (Asturias, Spain).Peer reviewed