Electrophoretic analysis of [3H]proline-labeled culture medium proteins of MCF-7 cells revealed the presence of disulfide-bonded, bacterial collagenase-sensitive component which comigrated with pro(alpha)1 chains of type III and type I collagens. However, it was pepsin- and trypsin-sensitive. Within 1 min of pepsin-digestion, a component with a size of alpha1 chain of type I or III collagen was produced which degraded after 5 min of digestion. Similarly, the pepsin-sensitive band was completely degraded by trypsin at 30 degrees C within 5 min. We examined CNBr peptides of the collagenous band and demonstrated that it was alpha1 chain of type III collagen. When MCF-7 cells were cultured in the presence of 2 nM estradiol, a marked increase in the level of collagen secreted into medium was found. The identified proteinase-sensitive type III-like collagen as major protein of extracellular matrix, would be expected to be more susceptible to degradation which might contribute to tumor progression.