The cDNA for human elongation factor-1 alpha was isolated from a cDNA library of human fibroblast cells. Using the cDNA as a probe, a number of chromosomal genes encoding the human elongation factor-1 alpha were isolated. Characterization of the clones by restriction enzyme mapping and nucleotide sequence analysis has revealed that only one of them is an active gene, whereas all of the other genes are processed pseudogenes. The active gene consists of 8 exons and 7 introns spanning about 3.5 kilobases, and the sequence of its exons is completely identical to that of the human elongation factor-1 alpha cDNA. The first non-coding exon of 33 base pairs is separated by a 943-base pair intron from the coding exons. The primer extension of human elongation factor-1 alpha mRNA has indicated that the transcription of human elongation factor-1 alpha gene starts from a C residue, and a "TATA" box was found 24 base pairs upstream of the initiation site. Three and five Sp1 binding sites are present on the 5'-flanking region and the 1st intron, respectively. Furthermore, one Ap-1 binding site is located in the 1st intron. By using nuclear extracts from HeLa cells, the promoter of human elongation factor-1 alpha gene could stimulate in vitro transcription better than the adenovirus major late promoter.