Using a rabbit model of lamellar keratoplasty (LKP), we examined the effect of standardized Barraquer cryolathe freeze injury on corneal structure and keratocyte biochemistry. Cryolathe corneal freeze injury caused immediate disruption of the stromal keratocytes and initiated an inflammatory cell reaction peaking at 3 days following freeze injury. Frozen rabbit LKP corneas were hazy in the early postoperative period, an appearance analogous to the postoperative appearance of human keratomileusis and keratophakia cases. The clinical appearance correlated with keratocyte death and increased interfiber collagen distance. Keratocyte regeneration occurred within 21 days. The regenerating “keratoblasts” were characterized by active mitochondria and an increase in the rough endoplasmic reticulum. Keratocyte counts in the central cornea were found to correlate significantly with corneal clarity. Collagen synthesis by regenerating keratocytes in frozen LKP corneas was characterized by a decrease in the proportion of type V collagen relative to type I collagen when compared to that synthesized by fresh LKP corneas. Although the collagen phenotypic expression of type I and type V collagen was unaltered, there was a change in the solubility characteristics of newly-synthesized collagen in frozen LKP corneas, with a significant decrease in the salt-solubilized collagen. This finding suggests that the collagen synthesized by regenerating keratocytes was more quickly incorporated into mature collagen forms. These studies suggest that freeze injury produced by cryolathe refractive surgical procedures induces a significant early effect on corneal structure and keratocyte biochemistry.