The aim of this study was to provide the biochemical and structural characterization of Piacentinu Ennese cheese and to evaluate the impact of different farm technologies on cheese proteolysis and microstructure. Fifteen cheeses were manufactured according to traditional technology, i.e., from raw milk and farmhouse rennet in the absence of starter culture. Pasteurized milk, commercial rennet, and starter were used for production of 20 nontraditional cheeses. Proteolysis in Piacentinu Ennese cheese was monitored during a 2- to 10-mo ripening time. Low rates of overall proteolysis were observed in cheese, as percentages of total N soluble at pH 4.6 and in 12% trichloroacetic acid were about 11.40 and 8.10%, respectively, after 10 mo of age. Patterns of primary proteolysis by urea-PAGE showed that alpha(s)-caseins were degraded to a larger extent than were beta-caseins, although a considerable amount of both caseins was still intact after 10 mo. Reversed phase-HPLC analysis of the cheese peptide fractions showed a slow decrease in the levels of hydrophobic peptides coupled to increasing levels of hydrophilic compounds as the cheese aged. The structural characteristics of Piacentinu Ennese cheese were evaluated by scanning electron microscopy after 2, 4, and 6 mo of age. The micrographs showed a sponge-like structural network with a well-distributed system of empty spaces, originally occupied by whey and fat. The microstructure changed during cheese ripening to become more compact with cavities of smaller size. Farm technology significantly affected cheese proteolysis and microstructure. Nontraditional cheeses had higher levels of pH 4.6-soluble N and showed a larger hydrolysis of alpha(s)-casein fractions by urea-PAGE analysis than did traditional cheeses. Large differences between cheese-types also concerned the patterns of secondary proteolysis. Nontraditional cheeses had higher levels of 12% trichloroacetic acid-soluble N and showed larger proportions of free amino acids and hydrophilic peptides in the HPLC profiles of the corresponding 70% ethanol-soluble N fraction than traditional cheeses. Nontraditional cheeses also had a more open structure with a coarser and less continuous appearance than did traditional cheeses. A large amount of variability in cheese proteolysis and structure within nontraditional treatment reflected farm-dependent changes in manufacturing conditions related to the use of various types of rennet and starter.