The four nuclear factor I genes (NFI-A, NFI-B, NFI-C, and NFI-X) give rise to multiple isoforms by alternative splicing in many tissues. These NFI proteins cooperate with AP-1, Myc, and other transcription factors in regulating transcription of numerous cellular and viral genes. We have investigated the growth-regulatory potential of NFI by overexpressing cDNAs from chicken NFI genes -A, -B, -C, and -X in chicken embryo fibroblasts (CEF). None of the NFI cDNAs induced oncogenic transformation of CEF. However, overexpression of each of the NFI proteins caused similar morphological alteration of the cells, inducing them to become flattened and polygonal and to show increased adherence. The growth properties of these cells were similar to normal CEF. When these morphologically altered CEF were challenged by superinfection with oncogenic retroviruses, they were resistant to transformation by the nuclear oncogenes jun, fos, junD, myc, and qin but were readily transformed by cytoplasmic oncogenes src, mil/raf, ras, and fps. The NFI-A1 protein was able to alter transactivation by the cellular and viral Jun proteins in a promoter-dependent manner. The changes in cell morphology and reduced susceptibility to nuclear oncogenes were not seen with a carboxy-terminal truncation in the transactivation domain of NFI, suggesting that this region of the protein is essential for the observed effects. The dichotomy between the activities of nuclear and of cytoplasmic oncogenes in this system is discussed.