The concentrations of L- and D-lactic acid and free fatty acids, C4:0 to C18:3, were quantified in a range of commercial enzyme-modified Cheddar cheeses. Lactic acid in Cheddar enzyme-modified cheeses varied markedly depending on the manufacturer. Differences in the ratio of L- to D-lactic acid indicate that cheeses of different age were used in their manufacture or contained varying levels of nonstarter lactic acid bacteria. The level of lipolysis in enzyme-modified cheese was higher than in natural Cheddar cheese; butyrate was the predominant free fatty acid. The addition of exogenous acetate, lactate, and butyrate was also indicated in some enzyme-modified cheeses and may be used to confer a specific flavor characteristic or reduce the pH of the product. Propionate was also found in some enzyme-modified cheese products and most likely originated from Swiss-type cheese used in their manufacture. Propionate is not normally associated with natural Cheddar cheese flavor; however, it may be important in the flavor and aroma of Cheddar enzyme-modified cheese. Levels of lipolysis and glycolysis appear to highly controlled as interbatch variability was generally low. Overall, the production of enzyme-modified Cheddar cheese involves manipulation of the end-products of glycolysis (lactate, propionate, and acetate) and lipolysis to generate products for specific applications.