High-level expression of a recombinant protein in Chinese hamster ovary (CHO) cells typically requires the laborious and time-consuming procedure of stepwise gene amplification. We hypothesized that use of transcription control regions from a highly expressed gene in CHO cells to drive expression of a gene of interest might reduce the requirement for gene amplification. To this end, we cloned a 19 kb DNA fragment containing the Chinese hamster elongation factor-1α (EF-1α) gene, as well as 12 kb of 5′ flanking sequence and 4 kb of 3′ flanking sequence. Expression vectors containing 5′ and 3′ flanking sequences from the Chinese hamster EF-1α (CHEF1) gene were constructed and, after insertion of six different reporter genes, transfected into CHO cells. For comparison, CHO cells were also transfected with the same six reporter genes inserted into commercial vectors utilizing either the immediate early promoter from cytomegalovirus (CMV) or the human EF-1α promoter. The striking result from these studies was that average expression levels from pooled, stable transfectants of CHEF1 vectors were 6- to 35-fold higher than expression levels from commercial vectors that utilize the CMV or the human EF-1α promoters. We also used a CHEF1 vector to express a secreted and a membrane-bound protein in stably transfected non-CHO cell lines. CHEF1-driven expression of secreted alkaline phosphatase (SEAP) in three of four cell lines tested (HEK 293, K562, L1.2, and HCT 116) was 13- to 280-fold greater than that from a commercial vector employing the CMV promoter. After transfection of four different cell lines of hematopoietic origin (K562, L1.2, JY, and Jurkat), the CHEF1 vector was found to express the chemokine receptor CCR4 at >10-fold higher levels than that driven from a commercial vector utilizing the CMV promoter. Results from these experiments suggest that the CHEF1 vectors will be useful for high-level protein expression not only in CHO cells, but also in a variety of other mammalian cell lines.