This investigation was conducted when previously repeatable experimental data became impossible to reproduce when using keratinocytes cultured in serum-free medium. Differences in calcium molarity between batches of medium were identified as a source of variation in cultured keratinocyte populations. The susceptibility of cultured keratinocytes to even small alterations in calcium molarity has been demonstrated. 2 regular medium batches were compared with a special preparation of medium, devoid of calcium chloride then supplemented with a known concentration of calcium ions. Culture progress in each medium was assessed by: morphological observation, % cells expressing involucrin and proliferating cell nuclear antigen, cell attachment, growth rate and colony forming efficiency. In order to control the phenotype of cultured keratinocytes, in a reproducible system, it is recommended that serum-free keratinocytes medium is purchased with the omission of calcium chloride. Supplementation of this medium may then be made by the investigator to suit individual culture requirements.