Phorbol ester-inducible phosphorylation of MARCKS, the '80-kDa' substrate of protein kinase C, was undetectable in several phenotypically dominant, non-transformed revertants independently derived from the ras-transformed cell line NIH3T3 DT-ras. Extremely low expression of MARCKS protein accounted for this apparent lack of phosphorylation. MARCKS-encoding mRNA levels were correspondingly decreased relative to normal and ras-transformed cells in all four ras revertant cell lines studied: C-11 and F-2, derived by 5-azacytidine treatment and selection with ouabain; CHP 9CJ, derived by ethylmethane sulfonate mutagenesis and selection with cis-hydroxy-L-proline; and 12-V3, derived by transfection with the human Krev-1 gene. However, re-expression of MARCKS after transfection of a cloned MARCKS cDNA into the C-11 ras revertant cells was not sufficient to induce retransformation. In fact, no significant difference in sensitivity to mitogenic stimulation by phorbol esters was observed among several cell lines expressing widely varying levels of MARCKS. This evidence argues against a direct role for MARCKS in mitogenic signaling. However, the strong correlation between attenuation of MARCIS expression and phenotypically dominant ras reversion suggests that a common negative regulatory mechanism might be responsible for both effects, presenting a potentially useful strategy for identifying factors involved in transducing the ras signal.