This study examined effects of repeated episodes of clinical mastitis in chronically infected quarters on milk N-acetyl-beta-D-glucosaminidase activity and duration of clinical signs. Milk samples were collected at each clinical onset from 49 chronic mastitis cases on a 1700-cow Michigan dairy farm. There were 49 first episodes of clinical mastitis, 49 second episodes, and 13 episodes of third or more. Agents isolated were Staphylococcus aureus (18.4%), Staphylococcus (7.3%), no growth (20.2%), environmental pathogens (streptococci other than agalactiae, Escherichia coli, Klebsiella spp., Enterobacter spp., and Citrobacter spp.) (22.0%), other pathogens (Serratia spp., Bacillus spp., diphtheroids [Corynebacterium spp. and Actinomyces pyogenes], Pseudomonas spp., and Nocardia spp.) (11.9%), mixed pathogens (two agents isolated) (12.8%), and contaminated samples (7.3%). Etiologic agents, duration of clinical signs, and NAGase did not differ by episode number. The correlation between log of NAGase and log of time until clinical recovery was .34. The relationship between NAGase and duration of clinical signs was strongest for second episodes, and weakest for third and greater episodes of chronic mastitis.