Previous studies suggested that transcription enhancer factor-1 (TEF-1) was involved in mediating the human chorionic somatomammotropin (hCS) gene enhancer (CSEn) function (Jiang, S.-W., and Eberhardt, N. L. (1994) J. Biol. Chem. 269, 10384-10392). We now show that an unrelated protein (CSEF-1) found in BeWo and COS-1 cells binds to the GT-IIC enhanson in CSEn and is correlated with CSEn activity in these cells. TEF-1 and CSEF-1 were distinguished by differential migration as GT-IIC complexes, thermal stability, molecular mass, and cross-reactivity with chicken TEF-1 antibodies. TEF-1 and CSEF-1 bound to the GT-IIC and Sph-I/Sph-II enhansons with identical binding properties, and in vitro generated TEF-1 competed with CSEF-1 binding to the GT-IIC motif, suggesting that their actions might be mutually exclusive. Up- and down-regulation of TEF-1 levels by expression systems and antisense oligonucleotides demonstrated that TEF-1 inhibited the hCS promoter in a manner independent of the enhancer or a known TEF-1 DNA binding site. The data suggest that TEF-1 may provide a counter-regulatory stimulus to the actions of CSEF-1, which may be involved in mediating enhancer stimulatory activity.