Occasionally, tumor cells differentiate spontaneously and then regress completely. It has been suggested that cAMP may be linked with the morphological differentiation of neoplastic cells since treatment of some tumor cells with dibutyryl cAMP, prostaglandin E1 and inhibitors of cAMP-phosphodiesterase induces irreversible morphorlogical differentiation. That this differentiation may be a reversion of malignancy is supported by the observation that no tumor is produced when these treated cells are inoculated into animals. Avian sarcoma virus-transformed mammalian cells also occasionally revert to a normal phenotype. Current information suggests four major categories of mechanisms by which transformed cells may revert to a normal phenotype: (1) loss of the viral genome; (2) mutation in the transforming gene(s) (by deletion, insertion or base change); (3) reduction in transforming-gene expression at either transcriptional, translational, or posttranslational levels; and (4) the appearance of host-cell resistance to the effects of viral transforming genes. To investigate factors that affect phenotypic reversion of transformed cells, we have chosen a cell line 433 of NIH 3T3 cells containing the transforming ras-gene of Harvey sarcoma virus flanked by LTR of MMTV; the expression of rasgene in 433 cells is therefore controlled by mouse mammary tumor virus promoter (MMTV-LTR) which is under control of glucocorticoid. Thus, the phenotypically normal 433 cells become transformed and produce the ras-gene product, p21 only upon addition of glucocorticoid. The goal of this study is to investigate the effect of intracellular regulatory factors, such as cyclic nucleotides, hormones, and growth factors on the controlling element, MMTV-LTR to gain knowledge on the mechanism of reverse transformation.