In vitro, Syrian hamster embryo (SHE) cell transformation is a neoplastic process that proceeds through several identifiable consecutive stages including in vitro, morphological transformation (mt), acquisition of immortality (I+), acquisition of tumorigenicity (T+) and tumor-derived cells (I TD). Eight transformed lineages consisting of cells at the mt, I+, T+ and I'TD stages were assayed for alterations in general markers of cell differentiation, mitogenic signaling pathways, cytoskeleton and cellular growth in 3D matrix. Alterations in cellular differentiation markers included a decrease in HI9, gene expression and placental alkaline phosphatase enzymatic activity at the mt stage in all lineages examined with a complete absence of HI9, gene expression and placental alkaline phosphatase enzymatic activity by the I'TD stage in a majority of transformed lineages. Changes in mitogenic signaling pathways included the production of autocrine growth factors and alterations in growth factor-induced immediate early gene expression by the I'TD stage of transformation in the majority of transformed lineages investigated. By the I'TD stage of transformation in most lineages, changes in both the cytoskeleton (including a decrease in tropomyosin-1 gene expression) and the Matrigel growth characteristics of SHE cells were observed.