Using a culture cell system, derived from a low-tumorigenic strain of L929 mouse fibroblasts, including cell variants with different malignant behavior in vivo, we have tried to dissociate a group of biological and biochemical transformation-associated properties with respect to the process of neoplastic progression. It is shown that morphologic changes, nonalignment in vitro, and growth in soft-agar are not sufficient alterations for in vivo malignant behavior and, also, not useful indicators for further neoplastic progression. The rate of hexose uptake should also be considered as unrelated to this process. The increase in tumorigenicity, but without the concomitant development of metastasis ability of the cells, was associated with the decrease of a 160k cell surface protein and the diminution of the tumor dose 50%. Finally, further alteration in morphology and the appearance of a 177k cell surface protein were associated with the development of metastasis ability, in an early stage; loss of fibronectin and the ability to grow as macrocolonies in increasing concentrations of deoxyglucose appeared as associated to more advanced stages of neoplastic progression. On the whole, the approach of taking the in vivo behavior, and not the in vitro properties, as end-point for the study of transformation is recommended on the basis of this and a previous paper.