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To determine if treatment with prostaglandin (PG) analogues affects keratocyte density as an indirect measure of extracellular matrix in the corneal stroma.In this case control study, 129 eyes from 68 patients were examined: 52 eyes had only PG analogue therapy for at least 3 years, 37 eyes had only β-adrenergic blocker therapy for at least 3 years, and 40 control eyes were without therapy. Scanning laser confocal microscopy was carried out, and each corneal stroma was subdivided into the anterior, midstroma, and posterior stroma. Keratocyte density was determined from the images by manual counting.Keratocyte densities in the entire stroma and in 2 stromal layers were significantly higher in patients with PG analogue therapy compared with control patients (entire stroma, P<0.001; anterior stroma, P=0.001; posterior stroma, P=0.016). Keratocyte density in the PG analogue therapy patients was also greater compared with the β-adrenergic blocker patients (entire stroma, P<0.001; anterior stroma, P<0.001; mid-stroma P=0.023, posterior stroma P=0.007). There were no significant differences between the control and β-adrenergic blocker groups. Overall, the density increase for the PG analogue group was greatest in the anterior stroma.PG analogue therapy increased the keratocyte density in each layer of corneal stroma, but more significantly in the anterior stroma than in the midstroma or the posterior stroma. The increased keratocyte density might be the result of diminished extracellular matrix, possibly owing to the known activation of matrix metalloproteinases and inhibition of tissue inhibitors of the metalloproteinases.