Aims: To determine the influence of cheese cooking temperature on autolysis and permeabilization of two lactococcal starter strains in broth and in Cheddar cheese juice during ripening. Methods and Results: Flow cytometry ( FCM) was used to identify and enumerate intact and permeabilized cells in broth and in Cheddar cheese juice. Levels of intracellular enzyme activities were quantified concurrently. Permeabilized cell numbers increased for both strains in broth following a temperature shift from 32 to 38 degrees C and was accompanied by an increase in the level of accessible intracellular enzyme activities. The relative proportions of intact and permeabilized cell populations, as detected by FCM in cheese juice, changed during 42- day ripening. Permeabilized cell populations increased during ripening for both strains; however, an increase in accessible intracellular enzyme activity was observed only for the highly autolytic strain Lactococcus lactis AM2. Conclusions: Differences in the autolytic and permeabilization response induced by cooking temperature in two lactococcal strains affects intracellular enzyme accessibility in Cheddar cheese. Significance and Impact of the Study: This study highlights the importance of the autolytic and permeabilization properties of lactic acid bacteria starter strains and their impact on cheese ripening.