A synthetic (Syn) medium was developed for growth of Micropolyspora faeni and Thermoactinomyces candidus, and optimum conditions for culture filtrate antigen production were found to be 3 days for T. candidus and 9 to 12 days for M. faeni. Appearance of proteolytic activity in the culture fluid supernatant coincided with a decrease in precipitating antigen content, protein content, and number of proteins on polyacrylamide gel electrophoresis (PAGE), probably as a result of proteolysis. The results suggest that protein content, number protein bands on PAGE, and proteolytic activity are predictive of precipitating antigen content. Antigens prepared in Syn medium were compared to those produced by the double-dialysis procedure and found to contain adequate amounts of antigenic material of sufficient quality to warrant clinical studies of their usefulness in the diagnosis of hypersensitivity pneumonitis.