The culture conditions of human keratinocytes were examined in serum-free medium with trypsin and/or trypsin inhibitor to achieve subculture operation in the successive passages of the cells. Although the cells inoculated in the trypsin-containing medium suffered serious suppression in the potentials of adhesion to the culture surface and subsequent growth in the early culture phase, the coexistence of trypsin inhibitor with trypsin in the medium resulted in a potential of cellular adhesion similar to that in the conventional medium without the treatment agents. Concerning the delay in cell growth, the cells treated with trypsin and its inhibitor showed a doubling time of 23 h, a comparable value to that of the untreated cells, in the exponential growth phase after exchanging the medium with a conventional one. This indicated that the potential of cell growth was restored by the medium exchange to remove trypsin and its inhibitor. In the cultures with trypsin and its inhibitor, it was found that adherent cell concentration at 96 h was maximized when time of first medium exchange (t(m)) was in the range of 24 to 36 h, and that prolonging t(m) caused an increase in lag time until cell division in a linear manner. The profile of cell growth was expressed by employing a cell placement model in the successive culture comprising the first and second passages, and the final cell concentration in the first passage and tm value in the second passage were determined so as to give an enhanced cell production rate under the examined conditions. Based on the calculated results, the successive culture was performed through subculture operation with the trypsin and trypsin inhibitor treatment, and the cell production rate obtained was at the same level as the culture associated with centrifugation.