We investigated the association between tissue elastolytic activity and the development of neointimal formation using a previously described porcine aortic organ culture. Neointimal formation is associated with the presence of intact endothelium (nondenuded cultures) but is markedly reduced if endothelial cells are removed (denuded cultures). In nondenuded organ cultures, elastolytic activity assessed by using [³H]elastin increased sixfold at day 3 after initiation of the culture (P<.01), a time earlier than the previously published increase in intimal smooth muscle cells (ISMCs). Elastolytic activity did not increase from day 3 to day 7 despite doubling of ISMCs but did double by day 14 (P<.01) and remained elevated to day 28, correlating with increases in ISMCs. In denuded organ cultures, elastolytic activity was much lower than in nondenuded organ cultures at day 3 (P<.05) but increased fivefold in the presence of nondenuded organ culture conditioned medium (P<.01). Addition of α₁-proteinase inhibitor for 14 days caused a 60% decrease in elastolytic activity in nondenuded organ cultures and a 27% reduction in ISMCs compared with untreated controls (P<.05 for both). The elastolytic activity, resolved as lytic bands on an elastin substrate gel, reflected candidate enzymes, one at 76 kD and perhaps a doublet at 43 and 50 kD. Our study suggests that endothelial cells release a soluble agent that enhances elastin-degrading activity in the aorta and may at least partially account for the initiation of neointimal formation.