Microbial enzymes within adjunct cultures are important for cheese ripening. Here, survival and proteolytic function of adjunct cultures of Lactobacillus plantarum strains MU12 and S6-4 on Cheddar cheese ripening were studied. Cheeses were ripened at 4°C, and samples were collected for analysis after 1, 30, 60, and 90 days. Lactococci numbers decreased by 2 to 3 log versus control, except in a few samples exhibiting significantly elevated numbers. Lactobacilli mainly originated from adjunct cultures, with lactobacilli numbers in adjunct-treated cheese significantly exceeding control numbers after day 30. Postripening, no significant differences were observed in composition (fat, protein, and moisture) and texture among cheeses, although observed significant differences in small nitrogen-containing compound levels (water-soluble nitrogen, trichloroacetic acid-soluble nitrogen, and phosphotungstic acid-soluble nitrogen) reflected proteolytic differences during ripening. Hydrolyzed protein, free amino acids, and volatile levels were consistently higher in adjunct-treated versus control cheeses and affected flavor. Cheddar cheeses may serve to effectively deliver beneficial organisms possessing proteolytic function.