The adaptation of normal human esophageal explants to organ culture for the first 33 d of in vitro growth was evaluated using histomorphology and [3H]TdR autoradiography combined with mitotic blockade. On the 3rd d in culture, extensive desquamation of superficial cells reduced the epithelium to about four cell layers. Thereafter, the epithelium remained atrophic, with a relative increase in basal and suprabasal cells. The percentage of cells synthesizing DNA was greatest from Day 4 through 8, just after desquamation, and reached a maximum on Day 4 (24 h [3H]TdR labeling index of 62%). The labeling index (LI) fluctuated, thereafter, but remained high (26% on Day 33). During the last 6 h of each [3H]TdR labeling interval, mitosis was blocked by colcemid. The 6 h mitotic rate (MR) was a reasonably constant fraction of the LI (maximum at 4 d: MR = 1.44%), but was much lower than predicted by [3H]TdR labeling indicating the loss of large numbers of cells after DNA synthesis but before or during mitosis. Unlabeled mitotic figures appeared between Days 1 to 3 and 6 to 33, suggesting that the epithelium initially contained a considerable population of cells arrested or delayed in G2 and continued to generate cells that remained in premitosis longer than 24 h. These results indicate that the atrophy observed in vitro is characterized by a relative increase in the basal and suprabasal cell category, a high replication rate, initial recruitment of cells arrested in premitosis, and rapid cell turnover with significant loss of cells at the premitotic or mitotic step, or both. Thus it seems that human esophageal epithelium grown in organ culture is a satisfactory substrate for experimentation (for example, in vitro carcinogenesis) that requires cell replication. However, there are major differences between the kinetics of esophageal epithelium in vivo and in vitro.