The population of stromal keratocytes represents a rarely examined topic. In the present paper we investigated the three-dimensional distribution of keratocytes in human donor corneas. The keratocyte density was calculated from biochemical measurements of the DNA content in samples of the corneal (and scleral) stroma obtained from well-defined regions. The DNA content of the central stroma (0-2 mm from apex) had a mean (+/- SD) of 1.25 +/- 0.30 micrograms DNA/mg dry tissue weight, corresponding to a cellularity of 4.6 +/- 1.1 x 10(4) cells/mm3 (n = 13). Towards the corneal periphery, the cellularity gradually increased to a 60-70% higher cell density at limbus. In the central stroma, an anterior-posterior cell gradient was found with a 30% lower cellularity in the subendothelial region compared to the subepithelial stroma (n = 8). In the four main quadrants of the central stroma, a uniform cell density was found while the peripheral areas showed a 10% higher cellularity in the superior quadrant. A close intraindividual correlation was observed between data obtained from 40 paired corneas ('cornea 1' versus 'cornea 2'); the stromal cellularity within 0-3.75 mm from apex (r = 0.81), the stromal cellularity within 3.75-5.5 mm (r = 0.92), the stromal cellularity within 5.5-8.0 mm (r = 0.85), and the endothelial cell density (r = 0.84). However, within a given cornea no correlation was found between the density of endothelial cells and keratocytes. These data define normal values for the regional density of keratocytes in the human cornea.