The ovalbumin gene (Ov) gene is responsive to estrogen, glucocorticoid, androgen, and progesterone. In our efforts to characterize the regulation of the Ov gene by steroid hormones, we have repeatedly observed that many vector backbones and promoters are responsive to steroids. In order to determine which vectors are most suitable for these types of analyses, vectors from Promega's pGL3 and newly engineered pGL4 Dual-Luciferase® Reporter Assay System were tested with both estrogen and/or corticosterone. The results confirmed that both series are induced by glucocorticoids in transient transfections of primary oviduct tubular gland cells, which contain glucocorticoid receptors, but not in MCF-7 cells, which do not. Modest effects that were dependent upon backbone and promoter context were observed with both series of vectors with estrogen. Thus, use of these vectors for experiments analyzing the effects of steroid hormones, especially glucocorticoids, should be done with caution. However, the new pGL4 series does have some advantages over the older series, and a comparison of transcription factor binding sites is reported.