The cellular mechanisms mediating intraocular pressure reduction following topical prostaglandin (PG) treatments are poorly understood. To determine if PG treatments might induce altered metabolism of extracellular matrix surrounding ciliary muscle cells, confluent human ciliary smooth muscle cell cultures were exposed to PGF 2α, 17-phenyltrinor-PGF2 2α, or 11-deoxy-PGE 1 for one to four days and the distributions of collagen types I, III and IV as well as laminin were determined immunocytochemically. In addition, collagen type 1V and promatrix metalloproteinase III (proMMP-3) content within treated cultures was determined using sandwich ELISAs. Compared with vehicle-treated cultures, there were substantial reductions in the density and branching of the collagen type IV-imnmnoreactive lattice accompanied by thickening of remaining strands in all PG-treated cultures. Similar changes were seen in the distribution of laminin within all PG-treated cultures. Reductions in collagen type III immunoreactivity were seen in cultures treated with either PGF 2α or 17-phenyltrinor-PGF 2α . No changes were observed in collagen type I immunoreactivity. Quantitative analyses revealed increased amounts of collagen type IV in both the culture medium and in extracts of the cell layer in all PG-treated cultures. In addition, there were substantial increases in the concentrations of proMMP-3 in all PG-treated cultures. These results indicate that PGs induce increased turnover and remodeling of ECM adjacent to ciliary muscle cells. Such changes may contribute to increased uveoscleral outflow in vivo following topical PG treatment.