The purpose of the studies described here was to define the biological behavior of the various clonally transformed colonies observed in cultures of carcinogen-exposed rat tracheal epithelial cells. As described in the preceding paper (H. Kitamura et al., Cancer Res., 46: 4631–4641, 1986), these colonies fall into four morphologically distinct categories. In the studies reported here we found that type I colonies had the smallest growth fraction (7%) and contained the lowest frequency of clonogenic cells (∼10−3). Colonies of types II to IV had mean growth fractions of 21 to 28%, and the frequency of clonogenic cells was 2 to 5 × 10−2 when measured under growth-permissive conditions (3T3 feeders). When the clonogenic cell assays were performed under selective conditions to identify cell variants which can grow without feeder support, the average frequency of such clonogenic cells in type I colonies was <4 × 10−5 and in colonies of types II to IV, between 5 × 10−4 and 10−2. In type IV colonies, the total number of cells per colony increased 8-fold between 5 and 12 wk postcarcinogen, but the clonogenic cell compartment increased 42-fold; the compartment of variant clonogenic cells, which are able to replicate on plastic, increased 139-fold during the same period of time. This indicated that major changes in the self-renewal capacity of the clonogenic cells were taking place during this early stage of transformation. Examination of the daughter colonies produced by replating colonies of types I to IV revealed that clonogenic cells with different growth potential existed within the same parent colony. Comparison of transformed colonies of the same type showed a marked degree of heterogeneity in the sizes of growth fractions and clonogenic cell fractions. These studies further indicated that, within all colonies, including the most advanced transformants, the majority of the cells were nonreplicating, terminal cells, suggesting that, at least during early stages of transformation, the transformed characteristics were not transferred from parent to daughter cells. With the exception of type I colonies, most of the colonies recognizable at 5 wk after carcinogen exposure progressed with time and acquired the morphological characteristics of type IV colonies, which were the most transformed phenotype. We conclude that transformation of rat tracheal epithelial cells is an asynchronous process and that the morphologically distinct types of rat tracheal epithelial cell colonies represent different stages of the clonal evolution of transformants.