We have studied the susceptibility of fibrils formed from fetal bovine skin type III collagen to proteolytic enzymes known to cleave within the helical portion of the molecule (vertebrate and microbial collagenase, polymorphonuclear elastase, trypsin, thermolysin) and to two general proteases of broad specificity (plasmin, Pronase). Fibrils reconstituted from neutral salt solutions, at 35°C, were highly resistant to nonspecific proteolysis by general proteases such as polymorphonuclear elastase, trypsin, and thermolysin but were rapidly dissolved by bacterial and vertebrate collagenases at rates of 12-45 mol x mol-1 x h-1. In solution, type III collagen was readily cleaved by each of the proteases (with the exception of plasmin), as well as by the true collagenases, although at different rates. Turnover numbers determined by viscometry at 35°C were: human collagenase, ≃1500 h-1; microbial (clostridial) collagenase, ≃100 h-1; and general proteases, 23-52 h-1. In addition, it was shown that pronase cleaves type III collagen in solution at 22°C by attacking the same Arg-Gly bond in the α1(III) chain as trypsin. However, like other proteases, Pronase was rather ineffective against fibrillar forms of type III collagen. It was also shown that transition of type III collagen as well as type I collagen to the fibrillar form resulted in a significant gain of triple helical thermostability as evidenced by a 6.8°C increase in denaturation temperature (T(m) = 40.2°C in solution; T(m) = 47.0°C in fibrils).