We examined the promoter activity of the 1.3-kb chicken β-actin gene sequence located between the 5' flanking region and the proximal region of the second exon. This promoter region showed higher promoter activity than the simian virus 40 (SV40) early promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) as assayed by transient lacZ gene expression in mouse L cells. Furthermore, replacement of the 3' splice sequence in this promoter by that derived from the rabbit β-globin gene resulted in a ~ 2.5-fold enhancement in the synthesis of β-galactosidase (βGal). Introduction of the SV40 origin of DNA replication (ori) into the vector carrying this hybrid promoter, which we designate the AG promoter, markedly enhanced the production of βGal in an SV40 T antigen-producing cell, BMT10. We have constructed a useful vector containing the strong AG promoter, several unique restriction sites, a SV40 polyadenylation signal and the SV40 ori for transient expression of cDNA in BMT10 or COS cells. We demonstrate the use of this vector for efficient production of interleukin-5 in BMT10 cells.