Purpose. To investigate whether exposure of human corneal epithelial cells to hyperosmotic stress activates the c-Jun NH2-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway, and stimulates production of the matrix metalloproteinases (MMPs): gelatinase (MMP-9), collagenases (MMP-1 and -13), and stromelysin (MMP-3). methods. Primary human corneal epithelial cells cultured in normal osmolar medium (312 mOsM) were exposed to media with higher osmolarity (350–500 mOsM) achieved by adding NaCl, with or without SB202190, an inhibitor of the JNK pathway; dexamethasone; or doxycycline for different lengths of time. The conditioned media were collected after 24 hours of exposure for zymography and ELISA. Total RNA was extracted from cultures treated for 6 hours and subjected to semiquantitative RT-PCR. Cells treated for 5 to 60 minutes were lysed in RIPA buffer and subjected to Western blot with phospho (p)-specific antibodies against p-JNK and p-c-Jun. JNK1 activation was also detected with an immunoassay system. results. The concentrations of MMP-9, -1, and -3 proteins in 24-hour conditioned media of corneal epithelial cells progressively increased as the media’s osmolarity was increased from 312 to 500 mOsM by the addition of NaCl. The concentration of MMP-13 progressively increased to a peak at 450 mOsM. Active p-JNK-1, p-JNK-2, and p-c-Jun were detected by Western blot as early as 5 minutes and peaked at 60 minutes in cells exposed to hyperosmolar media. The levels of p-JNK-1, p-JNK-2, and p-c-Jun correlated positively with the osmolarity of the culture media. The p-JNK inhibitor SB202190 and doxycycline markedly inhibited the stimulation of p-JNK-1, p-JNK-2, and p-c-Jun, as well as MMP-9, -1, -13, and -3 at both the mRNA and protein levels in the cells exposed to hyperosmolar media. conclusions. Expression and production of MMP-9, -1, -13, and -3 by human corneal epithelial cells correlated positively with increasing media osmolarity. This increase was mediated at least in part through activation of the JNK SAPK pathway. Doxycycline, an agent used to treat MMP-mediated ocular surface disease, inhibited the hyperosmolarity-induced MMP production and JNK activation. The relevance of these findings to stimulated production of MMPs by the elevated tear osmolarity in dry eye remains to be determined.