The CEF-4/9E3 gene is expressed constitutively in Rous sarcoma virus (RSV)-transformed cells. This expression is largely determined by an increase in transcription of the gene. In this report, we characterize the regulatory elements responsible for the transformation-dependent activation of CEF-4/9E3. Three sequences corresponding to AP-1, PRD II/kappa B, and TAACGCAATT are involved in the process and therefore define the src-responsive unit (SRU) of the CEF-4 promoter. In constructs containing a deletion of the SRU, multiple copies of AP-1 or PRD II/kappa B, but not TAACGCAATT, led to activation of the promoter. Thus, factors interacting with these elements are constitutively activated in RSV-transformed chicken embryo fibroblasts. In agreement with the results of transient expression assays, protein binding to AP-1, PRD II/kappa B, and TAACGCAATT were more abundant in the nuclei of transformed cells. The expression of the CEF-4 promoter was investigated in cells infected by a temperature-sensitive mutant of RSV. No significant increase in CEF-4 promoter activity was detected early after activation of pp60v-src. In contrast, a substantial activation of the CEF-4 promoter was detected late after a temperature shift. Factors interacting with the TAACGCAATT, PRD II/kappa B, and AP-1 elements accumulated gradually over a period of several hours. Therefore, transcriptional activation plays an important role in the late, constitutive expression of the CEF-4 gene in stably transformed cells.