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To assess the keratocyte density (KD) in the cornea of patients with keratoconus not using contact lenses.In this pilot study, the eyes (n = 68) of 35 patients with the diagnosis of keratoconus, who did not use contact lenses and the eyes (n = 70) of 35 healthy controls were prospectively examined using the Heidelberg Retinal Tomography Rostock cornea module (HRT3/RCM). The cases with keratoconus and controls were compared with regard to KD in the anteroposterior stromal layers.Of the cases with keratoconus, 15 (42.9%) were men and 20 (57.1%) were women. The mean age was 21.1 ± 11.1 (range = 14-41) years. Of the controls, 26 (73.9%) were men and 9 (26.1%) were women. The mean age was 23.9 ± 12.9 (range = 14-35) years. Of the 68 eyes with keratoconus, 22 (28.2%) had mild keratoconus, 23 (35.9%) had moderate keratoconus, and 23 (35.9%) had severe keratoconus. The mean anterior stromal KD was 651.4 ± 89 cells per square millimeter in cases with keratoconus and 879.4 ± 75 cells per square millimeter in controls (P < 0.05). The mean anterior stromal KD decreased by 25.9% in corneas with keratoconus. The mean posterior stromal KD was found to be 363.6 ± 74 cells per square millimeter in cases with keratoconus and 469.7 ± 56 cells per square millimeter in controls (P < 0.01). The mean posterior stromal KD decreased by 22.6% in corneas with keratoconus. The anterior stromal KD was higher than the posterior stromal KD (P < 0.001). No significant relationships were found between the stromal KD data and central corneal thickness or steepest keratometric.The KD in the anterior and posterior corneal stroma was decreased in patients with keratoconus, but without the use of contact lens, compared with healthy controls. This density difference was independent from the central corneal thickness and the stage of keratoconus.