The basic leucine zipper transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha) is involved in mitotic growth arrest and has been implicated as a human tumor suppressor in acute myeloid leukemia. We have previously shown that C/EBPalpha is abundantly expressed in mouse epidermal keratinocytes. In the current study, the expression of C/EBPalpha was evaluated in seven mouse skin squamous cell carcinoma (SCC) cell lines that contain oncogenic Ha-Ras. C/EBPalpha mRNA and protein levels were greatly diminished in all seven SCC cell lines compared with normal primary keratinocytes, whereas C/EBPbeta levels were not dramatically changed. Reexpression of C/EBPalpha in these SCC cell lines resulted in the inhibition in SCC cell proliferation. To determine whether the decrease in C/EBPalpha expression observed in the SCC cell lines also occurred in the carcinoma itself, immunohistochemical staining for C/EBPalpha in mouse skin SCCs was conducted. All 14 SCCs evaluated displayed negligible C/EBPalpha protein expression and normal C/EBPbeta levels compared with the epidermis and all 14 carcinomas contained mutant Ras. To determine whether oncogenic Ras is involved in the down-regulation of C/EBPalpha, BALB/MK2 keratinocytes were infected with a retrovirus containing Ras12V, and C/EBPalpha protein, mRNA and DNA binding levels were determined. Keratinocytes infected with the retrovirus containing oncogenic Ras12V displayed greatly diminished C/EBPalpha protein, mRNA and DNA binding levels. In addition, BALB/MK2 cells containing endogenous mutant Ras displayed diminished C/EBPalpha expression and the ectopic expression of a dominant-negative RasN17 partially restored C/EBPalpha levels in these cells. These results indicate that oncogenic Ras negatively regulates C/EBPalpha expression and the loss of C/EBPalpha expression may contribute to the development of skin SCCs.