To determine if keratinocytes influence melanocyte number and position in the developing epidermis we have experimentally recombined keratinocytes and melanocytes from epidermis of different stages of differentiation in the skin equivalent (SE) system. Previously we showed that developmental differences in the position and number of melanocytes characteristic of the epidermis in vivo were preserved in fetal and neonatal skin equivalents. In the present study we have combined cultured fetal or neonatal keratinocytes with age-matched or non-age-matched cultured melanocytes on the dermal equivalent. The ratio of basal keratinocytes to melanocytes (BK/M) present in multiple high-power fields was determined after localization of melanocytes by staining with the melanocyte-specific monoclonal antibody, HMB-45. The BK/M ratio in SE composed of neonatal keratinocytes and either fetal (n = 4) or neonatal (n = 5) melanocytes was 26.2 and 21.5, respectively. The BK/M ratio in SE composed of fetal keratinocytes and either fetal (n = 8) or neonatal (n = 5) melanocytes was 9.2 and 7.7, respectively. In each case, the BK/M ratio was dependent on the keratinocytes rather than the melanocytes. With either type of melanocyte, ratios in SE composed of neonatal keratinocytes were significantly greater than those with fetal keratinocytes. These results establish that keratinocytes regulate the BK/M ratio in this model and suggest that developmental differences between fetal and neonatal keratinocytes may be responsible for determining melanocyte numbers in the epidermal-melanin unit in vivo. The precise mechanisms that control the organization and number of melanocytes in the epidermis are unknown although keratinocytes may interact with melanocytes via growth factors, cell surface molecules, or other factors related to proliferation and differentiation of the epidermis