The prevalence of byssinosis in cotton mills is associated with proteolytic activity in cotton dust extracts. This study sought to characterize responsible enzymes. Proteolytic activities of saline and detergent extracts of: cardroom dust were assayed by hydrolysis of azocasein and synthetic substrates specific for trypsin, chymotrypsin and elastase. We assayed antiproteolytic activity of trypsin inhibition. Microbial growth was measured by serial dilution and transfer to trypticase soy broth. Saline extracts gave little or no proteolytic activity for the first 16 to 24 hours. The "early phase" was followed by a "late phase" of increased activity. Detergent extracts showed constant rates of hydrolysis for more than 24 hours before a late phase of increased activity. In both phases activity did not correlate with protein content of extracts. Microbial content increased by 10(6) at 26 hours; organisms hydrolyzed azocasein. Synthetic substrates are hydrolyzed at constant rates for 24 hours. Elastase substrate was hydrolyzed 2 times faster than tryptic substrate and 10 times faster than chymotryptic substrate. Antiproteolytic activity, present in most extracts, was inversely correlated with early phase proteolysis and directly correlated with protein content.