The inhibitory potency of four classes of compounds that inhibit corneal ulceration (thiols, tetracyclines, sodium citrate and sodium ascorbate) was assessed with collagenase purified from culture medium of alkali-burned rabbit corneas. The most potent inhibitor, a beta-mercaptomethyl tripeptide HSCH2(DL)CH[CH2CH(CH3)2]CO-Phe-Ala-NH2, exhibited 50% inhibition (IC50) at approximately 10 nM using the synthetic metalloproteinase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2. The inhibitor was somewhat less potent with type 1 collagen as substrate (IC50 between 1 and 3 microM), possibly because autooxidation of the essential - SH moiety of the inhibitor occurred during the longer time required for assay with the natural substrate. An N-carboxyalkyl tripeptide, CH3(CH2)2(DL)CH-(COOH)-Leu-Phe-Ala-NH2, was less potent (IC50 = 25 microM) than the thiol peptide. N-acetylcysteine, which is used to treat corneal ulceration, gave IC50 values of 2.7 mM and less than 10 mM with the synthetic and natural substrates, respectively. The IC50 values for the tetracyclines using the synthetic substrate were 15, 190 and 350 microM for doxycycline, minocycline and tetracycline, respectively. Inhibition by sodium citrate, but not the tetracyclines, could be reversed by excess Ca2+. Sodium ascorbate did not inhibit collagenase-mediated hydrolysis of either collagen or the synthetic substrate, thus indicating that the mechanism by which this agent inhibits corneal ulceration is not related to inhibition of collagen degradation by collagenase.