Human keratinocytes have been serially cultivated in low(0.015 mM) and high (1.8 mM) calcium containing medium. The calcium concentration of the growth medium significantly influenced the cell growth period in vitro. Cells grown in low calcium medium underwent 35-40 popu-lation doublings over 16-17 passages, while cells grown in high calcium medium ceased to proliferate after 20 population doublings over 7 passages. Changing the keratinocytes from one in vitro environment to the other drasti-cally altered the lifespan in culture of populations derived from the same primary tissue. The degree of DNA methylation of human keratinocytes was shown to decrease with age in both high and low calcium culture conditions but does not appear to be associated with differentiation.